Nuclear Magnetic Resonance Spectroscopy of Amino Acids, Peptides, and Proteins1

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY OF AMINO ACIDS. PEPTIDES. AND PROTEINS1

. .

By G . C K ROBERTS and OLEG JARDETZKY MRC Molecular Pharmacology Research Unit. Cambridge. England and Department of Pharmacology. Stanford University School of Medicine. Stanford. California

I . Introduction . . . . . . . . . . . . . I1. NMR of Amino Acids . . . . . . . . . . . A . Principal Features . . . . . . . . . . . B . Conformational Analysis . . . . . . . . . . C . Metal Ion Complexes . . . . . . . . . . D . Kinetics of Proton Exchange . . . . . . . . . LII . NMR of Peptides . . . . . . . . . . . . A . Primary Structure . . . . . . . . . . . . . . . . . . . . . . B . Conformation . C. Metal Complexes . . . . . . . . . . . D . Kinetics of Proton Exchange . . . . . . . . . . . . . IV . NMR of Polypeptides and the Helix-Coil Transition V . NMR of Proteins . . . . . . . . . . . . A . Assignment of Resonances . . . . . . . . . B . The Binding of Small Molecules to Proteins . . . . . 1. Studies of Ligand Spectra . . . . . . . . . a . Binding of Sulfonamides to Serum Albumin . . . . b . Binding of Choline Analogs to Antibody . . . . . c. Aspartate Transcarbamylase . . . . . . . . d . Lysozyme . . . . . . . . . . . e a-Chymotrypsin . . . . . . . . . . f . Binding to Proteins Containing Paramagnetic Metal Ions . 2 . Studies of Ligand and Protein Spectra . . . . . . a . Bovine Pancreatic Ribonuclease . . . . . . . b. Lysozyme . . . . . . . . . . . c. Staphylococcal Nuclease . . . . . . . . d . Binding of Cu(I1) to Ribonuclease . . . . . . C Protein Conformation and Conformational Changes . . . . 1 . Overall Conformation: Denaturation . . . . . . 2. Conformational Equilibria . . . . . . . . . 3 . “Heme-Heme Int.eractions” and Other Studies of Heme Proteins D . Probe Experiments . . . . . . . . . . .

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448 451 451 460 463 467 472 472 474 478 479 480 486 488 499 501 503 508 508 509 512 512 513 513 520 521 524 524 524 528 530 533

1 The preparation of this review was initiated a t t.he Department of Biophysics and Pharmacology. Merck Institute for Therapeutic Research. Rahway. New Jersey 07065. 447

448

G . C. K . ROBERTS AND OLEG JARDETZKY

VI. Concluding Remarks

.

Appendix: Basic Concepts References . . .

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535 536 54C

I. INTRODUCTION Over the past two decades high-resolution nuclear magnetic resonancf spectroscopy has become firmly established as the most powerful methoc for structure analysis in organic chemistry. Its application to structural problems in biochemistry has suffered an understandable lag, since thc molecular structures involved are of a higher order of complexity. The difficulties of both data collection and interpretation are therefore greater However, in the course of the past six years it has become apparent that these difficulties can be overcome. The types of problems that can bc formulated and solved with the aid of the method have become clearlj defined, and at least prototype experiments for most of the potential applications have been carried out. New though the method may be to biochemists, the scope of its usefulness has been clearly demonstrated. The contribution of a new method to an established field is properly judged by the new insights that it can provide. It therefore appears desirable for the purpose of the present review that the application of nuclear magnetic resonance (NMR) to protein chemistry be discussed primarily in terms of the information which the technique can contribute to the solution of the remaining problems of protein structure and function. The three principal questions to be examined are consequently the following: 1. What kind of information about proteins can be derived from NMR spectra? 2. What are the premises and procedures necessary to interpret the observations? 3. Is NMR merely another method for confirming conclusions reached by other methods (simpler or more cumbersome, as the case may be) or does it, in fact, permit findings not obtainable otherwise? For the initial examination of these questions, a very simple conception of the method itself will suffice. Some further details regarding basic concepts are developed in the Appendix. A more detailed introduction is given by Jardetzky and Jardetzky (1962), and comprehensive texts are available (Pople et al., 1959; Abragam, 1961; Emsley ef al., 1965). Nuclear magnetic resonance is a form of spectroscopy based on the absorption of radiofrequency electromagnetic radiation (-lo* Hz) by atomic nuclei placed on a strong external magnetic field (-104-106 oersted). As in other forms of spectroscopy, the absorption of a population of chemically identical particles appears as a bell-shaped curve on a plot of ampli-

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

449

tude us. frequency (or wavelength), with three principal measurable parameters: (1) intensity (given by the area under the absorption curve), (2) position of the line center (usually given in frequency units), and (3) width (usually measured a t half peak height, in frequency units). For the interpretation of these measurements in terms of the underlying atomic and molecular events it is useful to recall that the diameter of a nucleus is approximately lo6 times smaller than the diameter of an atom (10-14 us. 10-8 cm) and that the frequency of the radiation it absorbs is also approxinately lo6 times smaller than the frequency of ultraviolet or visible radiation that may be used to disrupt chemical bonds (lo8us. 10'4 sec-l). Because of the relationship between the frequency (v) and energy ( E ) of a quantum of electromagnetic radiation E = hv (where h is Planck's constant), this means that the energy changes involved in NMR absorption are very small even compared to the energies of thermal vibrations (lox2sec-I) which are observed in the infrared region of the spectrum. This permits us to regard the nucleus in a resonance experiment as a minute and essentially noninterfering monitor of the chemical structure and events in its surroundings. The very small energies involved in NMR absorption mean, however, that a relatively high concentration of nuclei is needed to observe a signal (2 M to observe a single proton on a single scan through the resonance). The detailed interpretation of the measurements is based on the following generally verified premises : 1. The intensity of a resonance absorption line is directly proportional to the number of nuclei in a given chemical environment and is independent of any other variables. This is a simple reflection of the fact that the nuclear magnetic moments responsible for the interaction with the electromagnetic field of the radiation are permanent and of fixed magnitude, in contrast to the a t least partially induced electronic transition moments responsible for absorption in the infrared, visible, and ultraviolet regions. 2. The position of a resonance absorption line is determined (a) by the density and geometric configuration of the electrons surrounding the absorbing nucleus and (b) by permanent local magnetic fields, such as those originating from other nuclei and unpaired electrons in close proximity (1-10 A) to the absorbing nucleus. The quantization of such local fields in the presence of the external field frequently leads to a splitting of resonance absorption lines, so that multiplets instead of singlets are observed for groups of chemically identical nuclei. Both the extreme sensitivity of line positions to differencesin the electronic environment and the additional information contained in splitting patterns account for the well known and widespread use of NMR for structure determination of organic compounds. 3. The width of resonance absorption lines is ultimately determined by

450

G . C. K . ROBERTS AND OLEG JARDETZKY

the rate of atomic motions. The theory is rather complicated in detail because internuclear distances and other factors enter into the equations, but it may be stat,ed in general terms that populations of nuclei in rapid motion-such as rotational diffusion of small molecules in liquids at room temperature, with periods of second-give sharp absorption lines (half-width Av55 = 0.1 Hz), whereas populations of nuclei in slow motionsuch as diffusion of macromolccules, with periods of second-give much broader absorption lines ( A q 5 = 10 to 1000 H a or more). Thus it is possible to (a) assign a given absorption line to a chemical group on the basis of line intensities and positions, (b) draw inferences about the electronic environment of the group from line positions and multiplicities, and (c) draw inferences about its mobility in space-including its exchange between two or more different environments-from line widths. More precise definitions of some of the terms used in describing N MR spectra are given in the Appendix. The foregoing outline is applicable to all forms of NMR spectroscopy regardless of the chemical naturc of the observed nucleus. Most of the following discussion, however, will deal more specifically with proton resonance, since the hydrogen nucleus is the most easily and hence most widely studied among the nuclei that could be used in the study of proteins, i.e., 'H, I3C, I5N, I7O, 31P,*H, 33S. In assessing the potential of NMR in protein chemistry, it is useful to bear in mind that the application of any physical method may be approached from two quite different points of view. In the first, a single variable or, a t most, a small number of variables is used to monitor a molecular species, a chemical transformation, or a structural change. Applications of thisth e monitoring or monoparametric-type constitute thc majority of all the familiar uses of physical methods in biochemistry: the use of ultraviolet absorption to determine protein concentration, fluorescence quenching to measure binding constants, light scattering to determine molecular weights, to name but a few. All methods that can provide a convenient and relevant parameter are of roughly equivalent value for this purpose. On the other hand, in the second, which we might call the multiparametric, type of application, thc aim is to use the sum total of the measurements possible by a given method to arrive at substantially more detailed conclusions concerning the object under study. Structure determination by X-ray diffraction is a prototype of this approach. The suitability of a given physical method for this type of application is clearly dependent on the information content of the measurements it permits, and even a cursory comparison of the existing methods leads to the conclusion that they are grossly imnequivalent from this point of view. There are several ways of assigning a numerical value to the information content of a physical method-particularly in spectroscopy. The simplest is to use the number of simultaneously measurable, independently inter-

N M R SPECTROSCOPY OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

451

pretable parameters obtainable from the spectrum of a given object. B y this criterion the information content of a high-resolution proton N M R spectrum of a small enzyme, for example, staphylococcal nuclease, may be set as 3 X 555 (3 parameters for each of the 855 proton resonance lines) = 2565, whereas the information content of its ultraviolet (UV) spectrum is a t best 3 X 11 = 33 (since the enzyme contains 11 aromatic residues). More refined estimates may yield somewhat different values, but the difference of two or more orders of magnitude between the information content of high-resolution NMR on the one hand and all other forms of spectroscopy on the other, remains. Both monoparametric and multiparametric applications of NMR t o protein chemistry have been described and will be discussed in subsequent sections. It is debatable whether any of the monoparametric uses of N M R have significant advantages over monoparametric measurements by other methods. The major importance of high resolution NMR in protein chemistry stems from the fact that the method permits, in principle, the simultaneous observation of all chemical groups comprising the protein. I t is thus the only existing method capable of providing very detailed structural information on proteins in solution. Before turning to the NMR spectroscopy of proteins, NMR studies of amino acids and peptides will be considered, since they not only provide valuable information about these molecules, but form the basis for the interpretation of protein NMR spectra. T o give a clearer idea of the kinds of information about peptides and proteins obtainable by NMR and of the procedures needed to obtain this information, an example of each major type of experiment will be discussed in some detail. I n general, the literature up to June 1969 has been surveyed comprehensively, though the coverage of the NMR of amino acids is more selective.

11. NMR

OF

AMINOACIDS

A detailed study of the NMR spectra of amino acids is of interest mainly because it is essential for the interpretation of protein N M R spectra. To be sure, such a study also provides a wealth of information about the structure of amino acids and their behavior in solution. However, a t this point NMR data for the most part only confirm conclusions already reached by other methods. We will therefore confine ourselves to a brief summary of the essential findings and place the emphasis of the discussion in this section on the few exceptions in which new insights have been possible. A . Principal Features The first high-resolution proton magnetic resonance spectra of amino acids were obtained by Takeda and Jardetzky (1957) and Jardetzky and

Ala

b h3

II

I I .

? ?

0 F

n

P

4

Leu 1.. 1

4.5

,

,

1

1

1

'

4.0

1

I .I '

I

1

1

1

3.5

1

1

'

1

1

3.0

1

'

1

1

1

2.5

1

1

'

1

1

2.0

1

1

II

I n 1

CHEMICAL SHIFT (ppm DOWNFIELD FROM DSS)

1

1

1.5

1

1

'

1

1

I.o

.

1

FIG.1. ( A X ) Nuclear magnetic resonance spectra of the common amino acids in the zwitterionic form (pH 5.5-6.0). The spectra were obtained a t 100 MHz, solutions of 0.2 M or less in D20being used. Intensities have been normalized to the largest single peak observed for a particular amino acid, and are thus not cornoarable from one amino acid t,o another (Arison. unniihlkherl ohnwvat,ions\

Ser

I

Thr

I

4.5

~

~

~

~

4.0

1

1

II

IIIII I,,

I I ~

1

1

3.5

1

l

1

l

1

l

1

~

1

1

1

1

~

1

3.0 2.5 2 .o 1.5 CHEMICAL SHIFT ( p p m DOWNFIELD FROM OSS 1

FIG.I(B)

1

1

~

~

1.0

1

1

1

1

1

1

1

1

1

~

454

G. C. K . ROBERTS AND OLEG JARDETZKY

lull

L.1

II

I

II Ill,.

I

I .

.I l l l l l l

I I

,,

I

CHEMICAL SHIFT (ppm DOWNFIELD FROM DSS)

FIO.l(C)

Jardetzky (1958) in D20 and water and by Bovey and Tiers (1959) in trifluoroacetic acid. The corresponding l3C spectra have recently been recorded by IIorsley and Sternlicht (1968). The principal features of these spectra, taking into account later refinements (Taddei and Pratt, 1964; Mandel, 1965; Nakamura, 1965; Bak et al., 1968), are summarized in Fig. 1. A comprehensive compilation of the available chemical shift and coupling constant data is given in Tables I and 11. The main conclusions of these studies are: 1. The chemical shift of the amino acid protons is dependent on the state of ionization of the amino acid. A plot of the chemical shift as a function of pH yields a titration curve, as shown in Fig. 2. 2. The change in electron density on titration, reflected in the chemical shift, is transmitted throughout the entire carbon chain in the aliphatic amino acids and the aliphatic portions of aromatic amino acids. The relative magnitude of the transmitted effect decreases with an increase in distance from the titrating group. 3. The effects of titrating groups in the side chain are similarly transmitted, as shown for histidine in Fig. 3. This allows a positive identification of the titrating group in, for example, the dicarboxylic or diamino acids (see Nakamura, 1965). 4. The magnitude of the coupling constants between protons on adjacent carbons is in some cases dependent on the state of ionization of the amino acid. This reflects differences in the preferred conformations in thc different ionized states. The detailed interpretation of this finding is, however, far from simple and is further discussed below.

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

5.0

-

n

,.

455

-

m A

"

A

CH

(alanine) P.

I

5

7I

9I

II I

1I3

I

PH

FIG. 2. Titration curve of amino acids in DzO. Chemical shifts are given with respect to benzene (7.20 ppm downfield from tetramethylsilane). Above pH -0.5 the NHa+ protons exchange rapidly with the solvent protons and are not observed as a separate resonance. From Jardetzky and Jardetzky (1962).

PH

FIG.3. Titration curve of histidine in DzO. The chemical shifts of the mCH, BCH2, and imidazole C2-H and C4-H resonances are shown relative to their values a t pH 5 1 (4.45, 3.2<5,8.70, and 7.49 ppm downfield from DSS, respectively), in HZ a t 60 MHz. From McDonald and Phillips (1963).

b aa TABLEI Chemical Shift" Data for the Common a-Amino Acids Amino acid Ma

A%

Cation (+) CaH

CBH

4.20 4.13

1.59 2.026

Asp

4.39

Asn

4.38

CYS

4.31

3.14

{z: :

3.06

3.47 j3.38

(CYS)z

4'55

Glu

4.16

Gln GlY

His

4.18 3.94 4.45

Ile

4.05

2.06

Leu

4.07

1.80

{i::: 2.27 3.49

Dipolar ion ( +)

Other CyH 1.88* CaH 3.27 -

CaH

CBH

Other

CaH

CBH

Other

?

4.107 3.21

1.467 1.63

C-yH 1.63 CBH 3.21 -

3.32 3.19

1.22 1.61

CyH 1.61 CISH 3.19 -

P

-

u1

3.010

-

Anion (-)

3'976

-

2.918 12.870 3.076 13.047 -

-

CyH 2.64

3.811

2.151

CyH 2.551

CyH2.55 ImC4H7.49 lmC2H 8.70 yCHz2.42 C6H 0.96 -yCH, 1.04 C-yH 1.80 CBH

3.768 3.548 3.984 3.655

2.140 3.191 3.136 1.95

3.72

1.70

C-yH 2.446 ImC4H 7.046 ImC2H 7.750 yCHz1.29 CISH 0.94 yCH, 0.996 CyH1.70 C6H 0.96, 0.94

3'56 3.59

3'56 3'58 3.23 3.27 3.22 3.51 3.10

3'27

2.64 {2,29 2.67 12.44 2.98 3.13 j2.92 1.84 1.86 2.97 12.84 1.64

(:62

-

P

zi

8M

2 +-

0

r CyH2.21 CyH2.31 ImC4H6.93 ImC2H 7.68 yCHz1.24b C6H 0.89 yCHa 0.93 CyH 1 , 6 8 CBH 0.90,0.92

8

+

k 3 z M

LYS

4.08

1.94b

Met

4.24

2.28

4.38

3'33 j3.26

Phe Pro

4.45

Ser Thr TrP

4'22 4.42 4.43

TYr

3.32

Val

3.98

ii:::

4.07 14.03 4.03 3.45 3.50 3.19 3.24 2.37

CyH 1.52b C6H 1.71 CcH 3.06 CyH2.69 SCHI 2.12

3.462

1.69

3.846

2.15

4H7.37

3.981

3.260 j3,134

CyH 2.06 C6H 3.42 -

4'11

2.11 12.31

3.84

3.95

CyH 1.36 ArC2H7.38 C4H7.73 C5,6H 7.28 C7H 7.59 Ar C3,5H 6.90 C2,6H 7.18

3.579 4.048

4.247 3.451 3.314

3.93

3.05 3.17

CyH 1.05, 1.07

3.595

2.268

C-yH 1.43 C6H 1.69 CsH 2.986 CyH2.968 SCHS2.126

3.24

1.53

3.23

l.Sb

3'46

i;:;;

CyH 1.72 C6H 2.74, 3.02 -

3.10 3.59

3.95 3.02 3.19

3.44

2.72 2.88

CyH1.20 Ar C2H 6.72 C4H7.26 C5,6H 6.72 C7H 7.02 Ar C3,5H 6.67 C2,6H 7.04

4H 7.370 CyH2.00 C6H 3.33,3.40 CyH 1.317 Ar C2H 7.307 C4H 7.720 C5,6H 7.279 C7H 7,524 Ar C3,5H 6.896 C2,6H 7.193 CyH 0.979, 1.330 ~

3.05

1.92

CyH 1.28 C6H 1.40 CeH 2.60 CyH2.56 SCHa 2.12

CyH 0.86, 0.92

All values are in parts per million downfield from 2,24imethylsilapentane 5-sulfonic acid (DDS); for tetramethylsilane (TMS), add 0.47 ppm; for hexamethyldisiloxane (HMS) add 0.33 ppm. Concentrations 0.2 M or less in DzO. The ionic forms (without taking (cation, pH 0.5), zk (zwitterion, pH 5.5-6.0), and - (anion, pH 12.5). All account of any charge on the side chain) are shown as figures quoted are those of Arison (unpublished); they are in general consistent with other, less complete, compilations (Taddei and Pratt, 1964; Nakamura, 1965; Mandel, 1965; Bak et al., 1968). Incompletely resolved magnetic nonequivalence of chemically equivalent protons. a

+

w M a

8

M

UJ

458

Dipolar ion ( k)

Cation (+)

Amino acid

J ~ B

Ma Arg

7.3 6.0

Asp

5.2

Asn

5.3

Glu Gln GlY

6.5 6.5

-

J88'

Other

-

J08

J88'

-

JBB,

-

7.3 -

-

6.4 6.4

-

Jar7.5 Ja7 7.5 Jq 0.3-0.6 524

4.0

Ja8

-

15.65

Ile*

Other

7.2-7.3 -

-

Anion (-)

0.9

-

14.7

Other -

-

J.5--,7.5-8.0

J4pO.7,0.2-0.4 Ju 1.5

G . C. X. ROBERTS AND OLEG JARDETZKY

TABLE I1 Coupling Constant" Data for the Common a-Amino Acids

Leub

6.0

-

LYS

6.3

-

Jg-,

Met6

6.1

-

J p - , 6.5

7.1 J-,a7.1 Ja. 7.0

6.2

-

5.75 16.65

Jg-, 7 . 3 J,a 7 . 3 J a . 7.2

-

Jg-, 7.0-7.5

14.4

3.5

Thr

3.7

Val

4.5

-

J ~ ~ 6 . 5 J,6 6 . 5 Jp, 7.0 J,a 7.0 Ja. 6.5 Jg, 7.5

-

13.4

-

-

J,a 6 . 5 J66,

12.1

-

-

11.8 -

Jp-, 6 . 6

15.6

15.5

J26

14.6

15.0

J A 8~. 9

-

6.5

14.7

15.0

Ser

-15

-

Jp, 7.0

4.4

-

0.7, 0.3-0.4

Jp, 6.9, 7 . 0

5.2

::: 3 ::;

{

5.0

5.0

11.2

14.5 14.0

-

10.5

Jgr 6 . 5 Jzo 0.75, 0 . 6

J A 8.5 ~ JB-,7 . 0

J,,, a In Hz. Conditions as for Table I. Spect.ra n o t amenabIe t o first-order analysis were analyzed using the program LAOCN3 (see Castellano and Bothner-By, 1964). Spectra could not be fully analyzed, as the number of spins involved exceeded the capacity of the program used.

-m

* 3 w

Ti

3

z

460

G. C. K. ROBERTS AND OLEG JARDETZKY

5. The zwitterionic form of the amino acids in neutral solution is directly demonstrable by a comparison of the area of the NH3' and the side chain protons, as readily seen in the early spectrum of alanine (Fig. 4). The observed proton chemical shifts can readily be correlated with the calculated electron densities on the corresponding carbon atoms, a,s shown by Del Re et al. (1963). A similar correlation has been established for the I3C shifts by Horsley and Sternlicht (1968). It should be emphasized, however, that chemical shifts are much more sensitive to variations in electron density than the methods of theoretical calculation used thus far.

Hz FIG.4. 40 MHz NMR spectrum of alanine in DtS04. The resonance lines are, in order of increasing field, (a) solvent, (b) NH,+, (c) aCH, (d) PCHa. From Jardetzky and Jardeteky (1962).

B. Conformational Analysis Among the problems of amino acid structure, the most difficult is the quest,ion of their conformation in solution. Among the physical methods available, high-resolution NMR is uniquely suited to the study of this problem. I t has been well known since the experimental studies of Lemieux et al. (1958) and the theoretical investigations of Karplus (1959) that the magnitude of the coupling constant between two protons on adjacent

NMR SPECTROSCOPY

OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

461

aliphatic carbon atoms is a function of the dihedral angle formed by the H-C1-C2 and C1-C,-H bond pairs.2 Although the equation originally derived by Karplus (1959) has not proved generally adequate, nevertheless when corrections for the electronegativity of adjacent substituents are introduced, a modified Karplus equation of the following form correlates most of the existing data:

J

=

A - B cos 4

+C

C O S ~4

- (D

+E

2

i=4

C O S ~3/2

4)

Aei

(1)

i=l

Where Aei = ei - 2.1, the difference between the electronegativities of the ith substituent and that of a hydrogen atom and k ; is an integer, k i = 1, 2 , 3 , 4 for the four possible substituents, taken in order of decreasing electronegativity. The set of coefficientsfound in this laboratory to be most suitable is A = 1.8, B = 2.5, C = 11.3, D = 0.4, E = 1.2. With these coefficients the accuracy of the calculated dihedral angle is within 5-10’, The value of the coupling constant characteristic of free rotation around a carbon-carbon bond (equal rotamer populations) is 6.9 Hz. The application of this relationship in principle allows the determination of rotamer

R

R

R

(1)

(2)

(3)

FIG.5 . Newman projections (viewed in the Cp -+ C , direction) of the three “staggered” rotamers about the C,-Cp bond of an amino acid.

populations about each C-C bond in an amino acid side chain, for each amino acid in a protein. Such a study has not yet been carried out, but when it becomes feasible, it will define the conformation of a protein in solution in unprecedented detail. A simplified procedure for the conformatjioiialanalysis of amino acids has been outlined by Pachler (1964). The conformation of an amino acid may be considered in terms of the relative populations of the three rotamers corresponding to the three energy minima about any C-C bond. Taking the C,-CB bond as an example, the rotamers are shown in Fig. 5.

* For a brief discussion of coupling constants, see the appendix a t the end of this review.

462

G. C . K. ROBERTS A N D OLEG JARDETZKY

I n many amino acids, the two @-protonsare magnetically rionequivalent (see Table I), indicating that the rotation about the C,-Cb bond is quite hindered. In these cases it is clear that the populations of the three rotameis about the C,-Cb bond are not equal. The observed coupling constant between the vicinal protons C,H and CbH will be a weighted average of the coupling constants in each of the rotamers, Jab = aJ1

+adz +

a3J3

(2)

where al, a2,and a3 are the relative lifetimes of each rotamer, so that a1

+ + a2

a3 =

1

(3)

and J1,J z , and JS are the coupling constants for this pair of protons in each rotamer. It is generally assumed, although thc validity of the assumption is uncertain, that the two gauche (g) coupling constants are the same, and the irans (t) coupling constant is different. If the p-group is a methylene with nonequivalent protons, one then has two simultaneous equations, in which the weighting factors and the respective gauche and trans coupling constants are the same, i.e., Jap1 =

aJg

Jab2 = a J t

+aJt +a J , + azJ, + a d ,

(4) (5)

From this one can obtain expressions for the relative lifetimes: a1

=

Jab2 Jt

- Jg -Jg

(6)

Thus, in principle one can calculate the lifetimes of each of the rotamers, provided that (1) one has reliable values for J t and J,; and (2) one can unequivocally assign Jabl and J,p to the individual methylcne protons. Given the relative lifetimes, the relative free energies of the rotamer pairs can be evaluated from the three relations

NMR SPECTROSCOPY OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

463

Much attention has been given to the evaluation of Jt and J,. The values calculated from the modified Karplus equation (1) are J , = 6.269 and J , = 2.844 for the zwitterionic form of an amino acid with a methyl substituent on Cg. Other estimates vary somewhat, but not enough to affect the values of the lifetimes by more than about 25%. Several sets of values of the relative rotamer populations in amino acids have been published (Pachler, 1964; Martin and Mathur, 1965; Cavanaugh, 1967). However, in none of these studies has an unequivocal assignment of the P-methylene protons been attempted. In the absence of such an assignment, Eqs. (4) and ( 5 ) can be written in an alternative form, equally compatible with the experiment, in which Jupl and J n p are interchanged. Under these circumstances the only rotamer that can be distinguished from the other two is rotamer 3, in which Jopl = Jugz = Jg. At present no method exists for unequivocally distinguishing between the P-methylene protons, short of stereospecific isotopic substitution of one of the protons by deuterium. Such experiments have not been done. It must therefore be borne in mind that all published values of rotamer populations in amino acids are subject to this inherent ambiguity. However, changes in coupling constants, for example with pH, must reflect changes in rotamer populations; thus it is qualitatively clear that the relative rotamer populations vary with pH as well as temperature. Examples will be found in Table 11.

C. Metal Ion Complexes The structure of metal-amino acid complexes in solution is another problem that can be studied by high-resolution NMR, since the formation of metal ion complexes results in characteristic changes in the spectrum of the ligand. The observed effects are quite different for diamagnetic and paramagnetic metals. The changes in chemical shifts produced by diamagnetic metal ions [such as Zn(II)] are comparatively small (0.2-0.4 ppm) and can be explained on the basis of charge effects. Thus, the binding of Zn2+to imidazole (Carlson and Brown, 1966) produces downfield shifts of the C2-H and C4,5-H peaks owing to the deshielding effect of the positive charges. The shift is not as great as that produced by the protonation of the imidazole ring, since the proton has a much higher charge density, and the aromatization of the

464

G. C. K. ROBERTS AND OLEG JARDETZKY

ring on protonation produces an additional downfield shift. Broadening effects are slight, if any. On the other hand, the effectsof paramagnetic ions on both the chemical shift and line width are much greater. The pronounced line broadening is due to the fact that the unpaired electron of a paramagnetic metal produces large local magnetic fields at all nuclei in its immediate vicinity. The fluctuation of these fields resulting from the random motion of the metal ion in the complex provides a very efficient relaxation mechanism for these nuclei. The degree of broadening is proportional to the inverse sixth power of the distance between the nucleus and the unpaired electron. Whenever the effect of a paramagnetic ion on more than one nucleus can be observed, it is therefore possible to define the position of the ion with respect to the ligand. Studies of this type have been carried out primarily with peptides (see Section III,C), but are equally feasible with amino acids. The broadening of ligand resonance lines is the only observed effect of a paramagnetic ion if the relaxation time of the unpaired electron, TI,, is relatively long. When TI, < 1/A (where A is the hyperfine interaction constant, related to the electron spin density at the appropriate nucleus), an additional phenomenon is observed for all nuclei connected to the ion by a system of bonding electrons. This is the ‘Lcontactshift.” Its magnitude depends on the number of chemical bonds between the observed nucleus and the metal ion. For nuclei removed by two or three bonds such shifts are often an order of magnitude greater than any seen in diamagnetic molecules (i.e., 10-20 ppm). Milner and Pratt (1962) have given a qualitative interpretation of the contact shift, and quantitative expressions have been derived by McConnell and Holm (1957); for a review see Eaton and Phillips (1965). The physical mechanism of the contact shift is as follows. Consider a 1 : 1 complex of Ni(I1) with ethylenediamine; the 3d orbitals of the nickel ion are split by the presence of an octahedral group of ligands into two groups-three t2, orbitals and two, higher energy, eg orbitals. The tegorbitals are fully occupied, while each eg orbital contains a single unpaired electron. These two electrons will be polarized in a magnetic field so that their spins will tend to point in the field direction. The Ni-N bonds lie along the same axes as the eg orbitals, so that there is a possibility of electron transfer from the nitrogen to the nickel; such transfer will preferentially involve the nitrogen electron in the Ni-N bond which has a spin antiparallel to that of the nickel e, electron. This results in a slight excess of electron spin density in the direction of the applied Jield at the nitrogen nucleus. This is referred to as positive spin density (negative spin density

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES,AND PROTEINS

465

being that opposing the applied field). Its effect is to increase the net magnetic field at the nitrogen nucleus and hence to decrease its shielding. By assuming that there can be a slight unpairing of the electrons in bonding orbitals, one can explain the transmission of spin density to other parts of the molecule through a-orbitals by a spin-polarization mechanism (transmission can also occur by electron delocalization through the 7r-orbitals of a conjugated system, if such a system is present). Thus the electrons associated with the hydrogen atom bonded to the nitrogen would adopt an antiparallel orientation, and this negative spin density produces increased shielding of the nucleus and an upfield shift of its NMR absorption. Similar arguments predict negative spin densities on the a-carbon atoms and positive spin densities on their hydrogen atoms; this is consistent with the observed shift to low field of the a-hydrogen resonance. At greater distances from the metal ion, the alternat,ion of chemical shifts along this chain appears to break down. A different kind of large chemical shift may also be observed in the presence of paramagnetic metal ions. It does not depend on a system of intervening bonding electrons and is referred to as the “pseudo-contact shift.” It is due to the residual magnetic field of the unpaired electron a t the observed nucleus, transmitted through space rather than the intervening electron system. This field is not averaged to zero whenever the electron is predominantly in an anisotropic orbital. The magnitude of the pseudocontact shift is inversely proportional to the cube of the distance between the observed nucleus and the unpaired electron. When a small molecule binds a paramagnetic metal ion, the shifts observed will be predominantly contact shifts; however, when the metal ion binds to a protein, a large number of nuclei not directly involved in the binding will be in close proximity to the metal ion, and a large number of pseudo-contact interactions is to be expected (see, e.g., McDonald and Phillips, 1969b). An example of the use of contact shifts to obtain structural information is the work of McDonald and Phillips (1963) on Co(I1)-histidine complexes. A t low pH (1.0-3.5), addition of Co(I1) to a solution of histidine produces marked downfield shifts of the (UGHand PCHs resonances, while those of the imidazole C2-H and C4-H are virtually unaffected. At pH < 1, there is no effect of Co(I1) on any of the histidine resonances, suggesting that the formation of the cobalt complex is associated with the ionization of the carboxyl group. The water resonance, which is shifted downfield in the presence of Co(II), is not shifted back toward its normal position to an appreciable extent, indicating that there has been little displacement of

466

G. C. K. ROBERTS AND OLEG JARDETZKY

D2O Complex I

D p Complex I1

Complex III

FIG.6 . Proposed structures for forir Co(I1)-histidine complexes. and Phillips (1963).

From M c l h n a l d

water from the first coordination sphere of the cobalt ion (complex I in Fig. 6). Sirice only single absorptions are scen for all the histidine protons, this complex must have a lifetime of less than about seconds. (Sce Section II,D for a discussion of the effects of exchange rates on NMR spectra.) As the pH of a solution containing histidine and Co(I1) is increased above 3.5, the NMR spectrum of histictine broadens and disappears, and the water resonance shifts toward that of Co(I1)-free water, indicating coniplete displacement of water from the coordination sphere of the ion. At pH 4.5 a spectrum with large contact shifts appears, and at pH 5 anothcr similar spectrum with somewhat larger contwt shifts is superimposed. These two spectra are clearly due to two different Co(I1)-histidine complexes, I1 and 111, which are exchanging relatively slowly with each other and with free histidine (exchange rates 5 6 x loBsec-I). The resonances of aCH arid PCH, in the spectra of these complexes were assigned by comparison with the spectra of the Co(I1) complexes of glycine and alanine; both these resonances are shifted downfield from their positioiia in

N M R SPECTROSCOPY OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

467

free histidine. Of the two imidazole ring protons, one is shifted upfield, and was tentatively assigned t o the C4-H, while the other is shifted downfield and was assigned to the C2-H. The spectrum of complex I1 is maximal in intensity a t pH 5.8 and disappears a t pH 7 ; that of complex I11 is maximal in the range pH 7-10.5. The effects of changing the Co(I1): histidine ratio in the relative intensity of the two spectra suggested that complex I1 has 1:1 stoichiometry, while complex I11 contains histidine and Co(I1) in a 2 : 1 ratio. The postulated structures are shown in Fig. 6 (11-111). At pH > 11.5, a new spectrum appears (that of complex I11 having broadened and disappeared by pH 11). This spectrum shows much smaller shifts of the resonances assigned t o C2-H, aCH, and PCH, (the last showing two peaks), but a larger shift of the C4-H resonance. Concomitant with thcse changes in the NMR spectrum, the color of the solution changes from pink to blue, and the mdgnetic susceptibility decreases from 5.1 t o 4.5 Bohr magnetons. This suggests a change in the geometry of the complcx from octahedral to tetrahedral, and the proposed structure is shown in Fig. 6 (IV). Similar Structures for the complexes of histidine with metals including Co(II), Zn(II), Cd(II), and Cu(I1) were suggested by Carlson and Brown (1966), based primarily on infrared studies but including some N M R results. Milner and Pratt (1962) have studied the complexes of Ni(I1) with amino acids in alkaline solution. They concluded that the bonding in the Ni(I1)histidine complex involved the imidazole N-1 and the NH2 groups; the structure is thus analogous t o complex (IV) of McDonald and Phillips (1963). Milner and l’ratt (1962) also noted that there were separate resonances for the two a-protons of the sarcosine (N-methylg1ycine)-Ni(I1) complex, while in the glycine-Ni(I1) complex there was a single aCH, resonance. They inferred that the chelate ring in the sarcosine complex is puckered, so that the two a-protons were nonequivalent, while that in the glycine complex is either planar or exchanging rapidly between two puckered conformations.

D. Kinetics of Proton Exchange Several phenomena in amino acid and protein chemistry involve the exchange of a proton, or of a group cont:iining 8,proton, between two or more magnetically nonequivalcnt environments. For example, protons attached to oxygen and nit.rogen atoms readily exchange between positions on the amino acid and 011 a solvent molecule. Similarly a carbon-bonded hydrogen, such as the &€I, may find itself in two or more environments, corresponding to diffcrent conformations of the molecule. If the rates of the exchange processes fall into a suitable range, they can be followed by observing either the resonance of the exchanging proton or the resoriance of a

468

G. C. K. ROBERTS AND OLEG JARDETZKY

neighboring nonexchanging proton to which it is coupled (see Jonas and Gutowsky, 1968; Binsch, 1968). The spectral changes that result from the exchange of a proton between two different environments fall into one of three different classes. If the lifetime, 7,in one of the environments, which determines the exchange rate, is long 011 the NMR time-scale [7 > 1 / ( 2 ~ A v ) ,where Av is the difference in chemical shift between the resonances of the proton in the two states], separate resonances for, e.g., the solvent H20 and the NH3+ group will be observed. This case is commonly referred to as the slow exchange case. If the exchange rate is faster (and the lifetime, 7, shorter), the two resonances broaden and coalesce a t 7 N 1/(2aAv), giving a picture characteristic of the intermediate exchange case. 111the rapid exchange case, when 7 << 1/(2~Av),a single sharp line is observed whose chemical shift is the weighted average of the shifts in the two states. Information about the rate of exchange can thus be obtained from the shape and line width of the NMR absorption of the exchanging proton. A similar set of three cases can be defined when the process is followed by observing the resonance of a nonexchanging proton coupled to the exchanging protons. The coupling will only produce a splitting of the resonance line of the noriexchanging protons if the exchange is slow. For example, the a C H absorption of the amino acids is split into a quartet by the NH3+ protons under conditions when the lifetime of a proton 011 the NH3+ group is long (T > l/J, where J is the coupling constant between the CYCHand NH3+ protons). As the exchange rate increases, the quartet coalesces to a broad singlet (at T ‘v l / J ) , which then becomes sharper (when T > l/J). It should be noted that the ranges for “fast,” and “slowJ1rates, defined by chemical shift differences and coupling constants are in general quite different (102-103 sec-’ for 6 as compared to 1-10 sec-’ for J ) . Sheinblatt (1962, 1963) and Sheinblatt and Gutowsky (1964) have used these effects to obtain information about the rates and mechanism of exchangc of the NH3+ protons of amino acids a t p H < 5. The most detailed information was obtained by Sheinblatt, and Gutowsky (1964) in their study of glycine and glycinemethyl ester. Under their experimental conditions, the resonance of the NH3f protons was too broad to be observed, so they derivcd values for the lifetime, T, of a proton on the NH3+ group from the line shape of thc aCH9 resonance. The observed line shape was compared with those calculated for various values of 7 from the equat,ions of Gutowsky et al. (1953). The value of 7 thus obtained is pseudo first order in the species, A, undergoing exchange, i.e.,

NMR SPECTROSCOPY OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

469

In the case of a bimolecular exchange of a proton between two species A and B,

so that 1

-

7

=

k[B]

(11)

This treatment can be extended to more complex mechanisms in a straightforward manner, and if several exchange processes are occurring simultaneously i

I n the following discussion of the results of Sheinblatt and Gutowsky (1964) the various ionic species of glycine wiIl be represented thus: R = HzNCHzCOOH R+ = H3+NCHzCOOH R* = H3fNCHnCOOR- = H2NCHXCOO-

For glycine methyl ester, only species equivalent to R and R+ exist. I n addition the following dissociation constants are defined

For pH < 1.8, the predominant species will be Rf, and the following possible mechanisms of proton exchange can be formulated.

+ HzO R + H30’ ki2 R+ + OH- C R + HzO k It + R+ It+ + R* R + It+ Rf + R

(I) R+

(11) (111)

(IV)

k’+l

ti

k+4

In the pH range under consideration, the concentration of OH- will be extremely small, and mechanism (11) can be ignored. Therefore, for glycine

470

G. C. K. ROBERTS AND OLEG JARDETZKY

and for the methyl ester 1 = k'+,[€I,O] T

+ k+l-K3[R+l [H+I

(Since the species R* cannot exist for the methyl ester, mechanism (111) Can be ignored.) These equations predict that 1 / should ~ be proportional to [R+]and to 1/[€1+]. In the pH range 0.6-1.8, this is in fact the case (see below), but for pH < 0.6, 1,'. is found to be indcpertdent of [R+],suggesting that mechanism (I) predominates in vcry acid solution. I n addition, I/. varies with l/[H+] in a nonlinear fashion. I n order to explain this nonlinearity, mechanism (I) must be modified as follows H

H

H (Ib)

I

-N----H-0

I

/

H

H

H ill

--+

H

I

-N

I

H

+ H20

€I

It is assumed that the formation of the NH3f - HZO hydrogen bond is extremely fast, and that proton exchange among water molecules is so fast that the reverse of reaction (Ib) has no effect on the observed rate. For reactions (Ia) and (Ib), assuming the concentration of the species

is constant, ~ k-l[H+], and for very acid solutions l c < li+lkn _1 -_ - = T

k-i[H+]

h'3kH

[H+I

(17)

The limiting slopes of the plot of 1 / us ~ l/[H+] in the very acid region are thus equal to K3kH. The values for ka obtained are, for glycine, 0.S5 10'0 sec-I, and for glycine methyl ester, 1.52 10'O sec-'. In the pH range 0.5-1.8, reactions (I), (111), and (IV) all appear to contribute. If the contribution of reaction (I) is given by Eq. (l(i), this can be simplified for kH > k_,[H+] to l / = ~ k+l. Therefore Eqs. (14) and (15) can be written for glycine

N M R SPECTROSCOPY

OF AMIKO ACIDS,

PEPTIDES, AND PROTEINS

471

and for glycine methyl ester

Thus I C + ~ can be obtained directly from the intercept of a plot of 1 / vs ~ l/[H+]; the values are, for glycine, 370 sec-' and for glycine methyl ester, 470 sec-1. Hence, given K3, k-1 is calculated to be 2.0 1O1O A4-1 see-I and 2.5 101" M - 1 sec-' rcspectively. I n the case of the methyl ester, the slope of this plot gives k+4 ( = 2.8 10'" Af-' seep1). If it is assumed that this value is the same for glycine, then, again from the slope of the line, k+3 = 1.2 l o 3 M-' sec-'. The lifetime of the water protons was also measured, from the broadening of the water resonance. Very accurate measurements were not possible, but approximate values for f , the fraction of NH3+ proton exchange events involving a water proton, were calculated. For glycine, f decreased from -1 a t pH 0.07 t o -0.9 a t pH 1.7, and for the ester it decreased from -1 a t pH 0.2 to -0.5 a t pH 2.2. This tends to confirm the suggestion t h a t mechanism (I) predominates a t very low pH, while mechanisms (111) and (IV) become significant as the pII increases. Between pH 2.7 and pH 4.7 the predominant species of glycine is the zwitterion, R*. For this case, the mechanisms of exchange considered were (I)-(IV) (with R+ replaced by R*) and also It*

(V)

+ R- k+a R- + R* k+e

(VU

R*$R

Thus the overall equation becomes

1 / was ~ found t o be a linear function of 1/[H+] a t constant [R*] in this pI-1 range. Furthermore the slopes of these lines, and their intercepts a t 1/[H+] = 0 were linear functions of [R*]. Thus the following numerical values could be obtained: k+j = 3.8 lo8M-I sec-'; Ic+z 5 2 10" M-l sec-'; k+l k+6 = 180 sec-'; k+g -k (k+*/K4) = 640 M-l sec-'. But

+

(zwitterion) k+, (cation)

k+l

-

Kz

N -

Ks

-

so that k+l (zwitterion) 5 sec-', and k+6 175 sec-'. I t should be emphasized that direct evidence for the separate existence of reactions (I) and (VIj or (111) and (IV) could not be obtained by this analysis.

472

G. C. K. ROBERTS AND OLEG JARDETZKY

111. N M R

OF

PEPTIDES

The NMR spectrum of a peptide or protein should, a t least to a first approximation, be the sum of the spectra of its constituent amino acids (Jardetzky and Jardetzky, 1957). The differences will be of two kinds: those due solely t o the inclusion of the amino acid in a peptide chain (primary structurc effects), and those that reflect the folding of the peptide chain and the conformation of the side chains (secondary and tertiary structure effects).

A . Primary Structure The effects of the primary structure on the aCH2 resonance of glycine have been analyzed in detail by Nakamura and Jardetzky (1967, 1968) using glycine-containing dipeptides and a series of oligoglycines. The aCH2 resonances of the oligoglycines were assigned unequivocally to particular residues by the synthesis of selectively deuterated oligoglycines. These assignments agreed with those of Li et al. (1962), and Sheinblatt (1966a), which were made on the basis of titration shifts, but differed from those of Mathur and Martin (1965). Nakamura and Jardetzky (1967, 1968) found that the chemical shift of the glycine aCH2 protons could be expressed as a simple sum of terms, each term representing the change in chemical shift on making a single chemical change. For example, formation of a peptide bond to the aNHz of a glycirie residue in the anionic form (so that no change in chargc occurs) causes a change in the chemical shift of the &HI, protons of about 0.66 ppm. This has been called the N-peptide shift. Formation of a C-peptide linkage or the titration of either the N or the C terminal groups will result in shifts of similar magnitude. The series of such shifts defined by Nakamura and Jardetzky (1967, 1968) is listed in Table 111. By adding together appropriate members of this series, it is possible t o predict the chemical shift of the glycine aCH2 protons in a peptide to within about 0.08 ppm. Most of the shifts listed in Table I11 are self-explanatory; the a-helix formation shift, which is clearly a function of the secondary rather than the primary structure, is included because its magnitude makes it a major determinant of the aCH chemical shift in polypeptides (sce Section IV). The so-called “correction” shifts are essentially the converse of the nearcst-neighbor titration shifts (see Table 111),arid arise because a group affecting the electron density of its neighbor does so a t the expense of its ow11 electron density. Its aCH resonance is thus shifted in the opposite direction. The N-terminal and C-terminal titration shifts are transmitted t o the side-chain protons in arnirio acids and t o the aCI-I of the neighboring residue in a peptide chain, but not beyond. On the other hand, the length

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

473

TABLEI11 Components of the Chemical Shift of the aCH2 Resonance in Glycine-Containing Peptidesa First-order effects 1. N-terminal titration shift =6(R)* - 6(R)- ‘v B(RX)* - 6(RX)-

=

0.45 f 0.01 ppm

2 . C-terminal titration shift A t c = 6(R)+ - 6(R)* ‘v 6(XR)+ - 6(XR)* = 0.3 f 0.01 ppm

3. N-peptide shift =

6(XR)-

- s(R)-

0.66

=

-

+ 0.01 ppm

4. a-Helix formation (see Section IV) AH = ~(R)I,,I~, - 6(Rh,

Second-order effects 5. C-peptide shift Apc = 6(RX)+- 6(1<)+

-0.3-0.6 ppm

= 0.00-0.02

ppm

6. N-nearest neighbor shift A P n ~ 6(YR)=

= +0.05-0.1

7. C-nearest neighbor shift AnC = S(RX)+ - 6(RY)-

ppm

= 0.0-0.2

ppm

8. N-nearest neighbor titration shift = G(XR)*

- 6(XR)-

‘v

0.05-0.1 ppm

9. C-nearest neighbor titration shift Atnc = 6(RX)+ - 8(RX)* = 0.05-0.1 ppm

10. Correction shift (see text)

a The shifts are defined as the difference in chemical shift (6) between two states, as given by the equations. The following symbols are used: R, The glycyl residue whose &H2 resonance is being measured. X,Y, Other amino acids. The sequences (e.g., RX) are given with the IT-terminal residue a t the left. +,- define the cationic, ewitterionic, or anionic state of the amino acid or peptide (charged side chains are neglected).

+,

of the peptide chain does not affect the magnitude of the peptide bond formation shifts or of the N-terminal and C-terminal titration shifts themselves. With the exception of the titration of the terminal aNH2 or CXCOOH,there appear to be no primary structure effects on the chemical shifts of the side-chain protons. It is to be expected that additivity rules similar to these will hold for all peptides, although the magnitude of the characteristic shifts will differ somewhat with the nature of the side chain. The experimental material

474

G. C. K. ROBERTS AND OLEG JARDETZKY

011 other peptides is still quite limited, but in general seems to bear out this expectation (Arison, unpublished data). The hope had been entertained that nearest-neighbor shifts, AnN and Ant, might be useful for the sequence analysis of peptides. The advantage of NMR over existing methods in this respect would have been that it is wholly nondestructive. However, there are major difficulties in the use of this method for peptides of even moderate size. The nature of an amino acid can be determined from the spectrum only by its side-chain resonances, not (with the exception of glycine) by its aCH resonance. Since the sidechain resonances are sensitive to the titration of the terminal amino and carboxyl groups but riot to other aspects of primary structure, only the N-terminal and C-terminal residues can be determined directly. Sheinblatt (1966b) has shown that it is possible to sequence di- and tripeptides in this way. The nearest-neighbor shift and nearest-neighbor titration shift of the trCH peaks should in principle make it possible to analyze longer peptides, if each aCH peak could be clearly associated with a group of side chain resonances by a decoupling experiment so that the nature of the amino acid could be determined. The difficulty in resolving the aCH and side chain peaks and particularly the difficulty in obtaining unambiguous assignments would, however, rise very rapidly with chain length. In addition, it is not possible to distinguish nearest-neighbor shifts from shifts produced by other residues, farther removed in the primary sequence, brought into close proxiniity by the folding of the peptide chain. Even the nearest-neighbor shifts in dipeptides are likely to depend upon the conformation of the side chains (particularly in the case of the aromatic amino acids). Sequence determination by NMR is thus likely to be restricted to very short peptides.

B. Conformation I n the spectra of the zwitterionic form of aminoacylglycines, the usual singlet aCHz peak is replaced by an AB quartet ( V A - Y B = 0.09-0.33 ppm; J A =~ 17.&17.6 Hz), indicating that the two aCHz protons are magnetically nonequivalent (Mandel, 1965; Morlino and Martin, 1967; Nakamura and Jardetzky, 1967). This nonequivalence is seen in other charge states or in glycylamino acids only when the second amino acid is aromatic. Two effects could contribute to this nonequivalence. The first is incomplete averaging of electric field gradients a t the a-carbon atom, even in the presence of free rotation (Pople, 1958). The second is restricted rotation leading to the existence of preferred rotaniers. Restriction of rotation in peptides is well known from the studies of Ramachandran et al. (1963). Nakamura and Jardetzky (1‘367) have shown that the restricted rotation about the Cal-C’ and N-Ca2 bonds in an aniinoacylglycine defines a limited

N M R SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

475

range of allowed conformations in which each of the glycine a-protons can be cis (with respect to the plane of the peptide bond) to either the terminal amino group or the side chain on CaI. The available range of “cis” conformations is quite different for the two glycine a-protons in aminoacylglycines, explaining their nonequivalence. I n the zwitterionic form of the dipeptide, rotamers in which the positive and negative charges are cis to each other will be favored, thus further restricting the freedom of rotation and increasing the nonequivalence of the two a-protons. It appears th a t in the presence of a magnetically anisotropic side chain, such as an aromatic ring, the nonequivalence is great enough to be observed even in the absence of the further restriction imposed by the charge-charge interaction. Bystrov et al. (1969) have studied the NMR spectra of a number of DD- and D1.-alanylalanine derivatives. Measurements of J N H - c H indicated that a c i s orientation of these tivo protons was predominant in nonpolar solvents (CDC13) (from infrared measurements, this was said to be a hydrogen-bonded cyclic structure). In more polar solvents (dimethyl sulfoxide or water) the relative population of this rotamer decreased. The use of NMR in the conforniational analysis of small peptides is well illustrated by the work of Stern et al. (1968) on gramicidin S. The NMR spectra of this cyclic decapeptide (sequence L(L-Val-LPro-D-Phe-L-Leu-L-orn)~~) in deuteromethanol and in dimethyl sulfoxide are shown in Fig. 7. The first problem in the analysis of such a spectrum is the assignment of the various peptide ITH, aCII, and side-chain resonances to particular amino acids in the molecule. Stern et al. (1968) used double-resonance techniques to demonstrate which resonances were spin-spin coupled to each other. I n this way, they could group the resonances as shown in Table IV, and by the symbols in Fig. 7. These groups of resonances were TABLEIV

Coupled Resonances i n the N M R Spectrum of Gramicidin So

Chemical shifts (ppm from T M P )

b

NH

(UCII

SCHz

Assignment

7.7 8.9 8.8 8.7

4.2 4.5 4.7 4.95

3 0 1.5

Valirie Phenylalaiiirie Leucine Ornithine

Stern et a!. (1968). TILTS, tetramethylsilane.

476

G. C. K. ROBERTS AND OLEG JARDETZKY

r\2i +

L

FIG.7. 100 MHz N M R spectra of gramicidin S in dimethyl sulfoxide-ds (DMSO-ds) (upper) and CDIOD (lower). The chemical shift scale is in parts per million downfield X, and indicate regions confrom tetramethylsilane (TMS). The symbols 0 , 0, nected by decoupling. The large resoriarice a t 4.8 ppm in the lower spectrum is due to water impurity. From Stern et al. (1968).

+

assigned to specific residues by making use of the characteristic features of the spectrum of each amino acid; for example, the PCH, resonance of phenylalariirie occurs a t about 3.0 ppm, further downfield than the PCHz resonances of the other amino acids present in gramicidin S (see Table I), so the group of resonances a t 3.0, 4.5, and 8.9 ppm (and the phenyl resonance a t 7.3 ppm) can be assigned to phenylalanine. These assignments differ in some respects from those previously made by Liquori arid Conti (1968). However, they have been confirmed by Conti (1969). It is apparent from the work of Stern et al. (1968) that there is only a single set of resonances for each pair of identical amino acids and therefore that the members of each pair are in identical environments; this suggests that the molecule has Cfvsymmetry. Second, the coupling constants between the &H and the peptide XH, J N C , are similar (8.5-9.0 Hz) for the ornithine, leucine, and valine residues, but much smaller (in fact unresolved) for the phenylalanine residues. This coupling constant is sensitive to the confor-

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

477

mation about the N-C, bond, and comparison with literature values suggested that large values of JNCindicate that the N-H and C,-H bonds are oriented trans with respect to the N-C, bond, while small values of J N C indicate a cis orientation. On this basis, Stern et al. (1968) concluded that the dihedral angle about the N-C, bond, 4, had a value of about 30" in the ornithine, leucine, and valine residues, and of about 150" in the phenylalanine residues. Finally, the exchange rates of the amide protons were determined in CDBOD and in DMSO 5% DzO. They fell into two classes: the amide protons of the ornithine and phenylalanine residues exchanged fairly rapidly (<24 hours), while those of valine and leucine exchanged much more slowly ( > 1 week). Stern et al. (1968), concluded from this that the amide protons of valine and leucine were involved in hydrogen bonds. They proposed a structure in which the two tripeptides L-Val-L-Orn-L-Leu formed an antiparallel pleated sheet, with the hydrogen bonds involving the NH and carbonyl groups of the valine and leucine residues. These two tripeptides (in which the dihedral angle, 4, was set at 30") could readily be joined a t both ends by D-Phe-L-Pro sequences, with 4 = 150" for the phenylalanine residue. It should be noted that no information about the proline residues was obtained from the spectra; in the model, the proline rings were almost a t right angles to the plane of the pleated sheet structure. Hydrogen-bonding is by no means a unique explanation for the slow exchange of the valine and leucine amide protons in this molecule, and steric hindrance is perhaps almost as likely. In addition, the valine amide proton resonance is farther upfield than the other amide protons-the opposite shift t o that which would be expected if this proton were involved in a hydrogen bond [Stern et al. (1968) attribute this upfield shift to the anisotropy of a nearby carbonyl group]. It seenis that the direct evidence for hydrogen bonding to the valine and leucine amide protons is rather weak. The proposed structure is, however, very similar to those suggested by Hodgkin and Oughton (1957) and Schwyzer (1958), and further evidence for it has been advanced by Schwyzer and Liidescher (1968). They have prepared a derivative of gramicidin S in which the 6NH2 groups of the ornithine residues are substituted with phthaloyl groups. I n the XMR spectrum of this derivative, the peaks of the two types of phthaloyl protoris were shifted upfield 0.1 and 0.2 ppm from their positions in N-phthaloyl glycine methyl ester. The single phenyl peak a t 7.25 ppm was split into two, at 7.24 ppm (six protons) and 7.16 ppm (four protons). Schwyzer and Ludescher (1968) attributed these changes to an interaction between the phthaloyl and phenyl rings and showed that the antiparallel pleated sheet model can explain this interaction, while other models (Liquori and Conti,

+

478

G. C. K. ROBERTS AND OLEG JARDETZKY

1968; Vanderkooi et al., 1966) cannot. Additional examples of conformational analysis of cyclic peptides by NMR have been described by Kopple and Marr (1967) and Kopple and Ohnishi (1969). The formation of complexes between the depsipeptide valinomycin and K+ or Csf ions has also been studied by NMR. Haynes et al. (1969) stated that the exchange rate between the free and bound forms of valinomycin in the Kf-valinomycin complex is slow (less than 0.2 sec-I). Unfortunately, it appears to us that this statement is not borne out by the published spectra. A more detailed study of the conformation of valinomycin and its potassium complex by NMR and infrared spectroscopy which has led to proposed structures for free valinomycin and the complex has been reported by Ivanov et al. (1969).

C . Metal Complexes Li et al. (1961, 1962) and Sheinblatt (1967) have studied the effects of Zn(II), Mg(II), Cd(II), and Cu(I1) on the N M R spectra of a number of glycylamino acids and oligoglycines in the anionic form. I n all cases the effect of adding the metal ion was greatest on the aCH2 resonance of the K-terminal glycine residue. This tends to support the conclusions of Li et al. (1957), from other data, that the m e t d ions are bound to the terminal amino group and the adjacent peptide nitrogen. A more detailed study of Ni(I1) and Cu(I1) complexes of oligoglycines has been reported by Kim and Martell (1969). I n general, their observations’confirm those of Li and co-workers (1961,1962), in that the effect of the metal ion is usually greatest o n the N-terminal residue. However, on addition of Ni(I1) to the zwitterionic form of the oligoglycines, Kim and Martell (1969) found that the broadening of the aCH2 peak of the C-terminal residue was greater than that of the N-terminal residue. Kim and Martell (1969) interpreted this t o indicate complex formation by the carboxylate group under conditions where the peptide nitrogens and the aNH3+ group were not dissociated, and thus bound the metal ion relatively weakly. I n alkali, when the peptide nitrogens are negatively charged, the Ni(I1) complex of triglycine or tetraglycine is diamagnetic and gives sharp resonance lines (Mathur and Martin, 1965; Kim and Martell, 1969); it appears that under these conditions the chelate has planar or tetragonal rather than the usual octahedral geometry. Falk el al. (1967) studied the complexes between Cu(I1) and triglycylglycine by electron spin resonance (EPR), potentiometric titration and proton relaxation rate measurements. The enhancement of the relaxation rate of the water protons (see Cohii, 1967; Mildvan and Cohn, 1969) in the presence of Cu(I1) or Cu(II)-triglycylglycirie was used to determine the hydration of the complexes. Cu(I1) binding to ribonuclease S-peptide has been studied by Bersohn and Ihnat (1968).

479

N M R SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

D. Kinetics of Proton Exchange Using similar methods to those described above (Section II,D), Sheinblatt (1965, 1966a,b) has studied the exchange of the peptide hydrogens of glycylglycine and glycylglycylglycine (triglycine) . The two mechanisms considered are:

+ HzO : RCONR’ + H80+ RCONHR’ + OHRCONR‘ + H20 k+l

RCONHR’

(I)

k+z

(11)

The values obtained for the rate constants and Ic+z in the pH range 7-9 are given in Table V; it appears that the exchange proceeds predominantly by mechanism (11) under these conditions. It is notable that, according to these figures, the rates of exchange of the two peptide hydrogens of triglycine differ by almost three orders of magnitude in the anionic form of the peptide. To explain these very different rates, Sheinblatt (l965,1966a,b) proposed that the rate of exchange of the peptide hydrogens was enhanced by the presence of a nearby positive charge or a second amide group, and inhibited by the presence of a negative charge. It seems unlikely, however, that titration of the terminal amino group of triglycine should produce a 10-fold reduction in the rate of exchange of NH(2), without any effect on X H ( l ) , which is much closer to the N-terminus. The chemical shift of the aCHz resonance of the C-terminal residue in triglycine is identical in the zwitterionic and anionic forms (Nakamura and Jardetzky, 1968), so that titration of the a-amino group produces very little change, if any, in electron density in the C-terminal part of the molecule. If a spectrum of the zwitterion of triglycine is run in D20, when all the peptide hydrogens will have been exchanged for deuterium, the line width of the aCH2 resonance of the C-terminal residue is appreciably greater than that of the other TABLEV Rate Constants for the Exchange of Peptide (Amide) Hydrogens ~~

N-Methyl acetamidea Glycylglycimeb

Rate constant k+l[HzO] (sec-I) k+,(M-I sec-l)

5.2

x

NH(1)

Berger et al. (1959). Sheinblatt (1965). Sheinblatt (1966b).

7.8x

NH(2)* -

5

3.5

106

108

~~

Triglycinec

6.7

x

109

8.4

x

H~XCH~CONHCHZCONHCH~COOH; f,zwitterion; -, anion, (1)

(2)

d

NH(2)-

107

0 . 8 x 107

480

G. C. K. ROBERTS AND OLEG JARDETZKY

two aCHz peaks (Nakamura and Jardetzky, unpublished). This indicates t h a t there are effects on the lineshape of this resonance other than the exchange of the neighboring peptide hydrogen (e.g., incipient nonequivalence of these two aCHz protons), and raises the possibility that the exchange rate of NH(2) in the zwitterion measured by Sheinblatt (196613) is in error. If the true rate of exchange of NH(2) for both the zwitterion and the anion is 8 X lo6M-' sec-', then all the data can indeed be rationalized by assuming an increase in the exchange rate by a factor of 300 in the presence of a neighboring positive charge, a further increase by a factor of about 3 in the presence of a second amide group, and a decrease by a factor of about 3 due to a neighboring negative charge.

IV. NMR

OF

POLYPEPTIDES AND THE HELIX-COILTRANSITION

As already indicated, in the absence of any secondary or tertiary structure the NMR spectrum of a polypeptide is a superposition of the spectra of its constituent amino acids, provided the shift of the a-proton is corrected for the effects of the formation of the peptide linkage, as outlined in Section II1,A. This makes it possible to detect secondary or tertiary

97.5 % CDCI,

A 500

A 400

300

ZOO

L 100

0

cps f r o m H M S

FIG. 8. NMR spectra (60 MHz) of poly-7-benzyl-L-glutamate DP340 (0.08 M residue concentration) at three different CDC13-lrifluoroaceticacid solvent compositions. From Markley et al. (1967).

NMR SPECTROSCOPY OF AMINO ACIDS,

-2 u

I

510 -

30 20

PEPTIDES, AND PROTEINS

I

-

481

-

Q-CH

structure by deviations from the expected spectrum. If one wishes to distinguish between secondary and tertiary structure effects, the study of models with a pure secondary structure, such as the or-helices of polypeptides, is useful. Many simple polypeptides, usually amino acid homopolymers, undergo a conformational transition from a random coil to a highly ordered or-helix under specific conditions of solvent composition and temperature. This transition has been studied extensively by optical methods. It can also be easily followed by NMR. The first XMR spectra of a polypeptide in both the random coil and helical conformations were obtained by Bovey et al. (1959). They found that the spectrum of poly-y-benzyl-L-glutamate in the helical conformation (in trichloroethylene containing 10% trifluoroacetic acid) is almost as sharp as that in the random coil (in trifluoroacetic acid). I n pure trichloroethylene, however, the spectrum disappeared completely; this was attributed t o

482

G. C. K. ROBERTS AND OLEG JARDETZRY

the aggregation of helices. However, the tumbling of even a single long, rigid helical rod would be slow enough to lead to incomplete averaging of dipole-dipole interactions, and therefore t o very broad or unobservable lines. The finding that the resonances of polypeptides in the helical conformation are only about 3-fold broader than those in the random coil (see, e.g., Markley et aE., 1967) suggests, therefore, t h a t the individual residues in the polypeptide chain exchange between the helical and coil states even when the overall helix content is high. Goodman and Masuda (1964) and Marlborough et al. (1965) havc described the relative broadening of the various resonances on helix formation. The changes in chemical shift that occur have been examined in detail by Marliley et al. (1967), Stewart et al. (1967), and Bradbury and co-workers (1967a,b 1968a,b). Typical spectra (of poly-7-benzyl-L-glutamate) are shown in Fig. 8, and the changes in chemical shift and line width are plotted in Fig. 9. Several generalizations can be made about the spectral changes that accompany helix formation in polypeptides. 1. The aCH peak shows an upfield shift on helix formation, amounting to 0.3-0.6 ppm, though in some cases the broadening of the peak is such that its chemical shift cannot be followed throughout the transition. This shift is most probably a result of the anisotropy of the pcptide bond. I n a right-handed helix of an L-amino acid the proton on the a-carbon is almost entirely above the plane of the peptide linkage preceding it (on the amino terminal side) and almost in the plane of the following peptide linlrage. I n this configuration, the x-electron system of the peptide bond preceding the a-carbon would produce an upfield shift of the aCH resonance, while that of the following peptide bond would produce a downfield shift. Since the shift is in fact upfield, the effect of the peptide bond on the aminoterminal side appears t o predominate. In a left-handed helix of an L-amino acid (poly-@-methyl aspartate; Katchalski et aZ., 1964) the aCH is in the plane of the peptide linkage preceding it and above the plane of that following. Again the shift is upfield; the shielding effect, in this case due t o the following peptide bond, is once again dominant. I n many such model systems the helix-to-coil transition is produced by addition of trifluoroacetic acid to a solution of the polypeptide in chloroform. The possibility therefore exists that part of the difference in the chemical shift of the aCH resonance between the two conformations is due to a solvation effect of the trifluoroacetic acid. However, Bradbury et al. (1968a) have shown that the chemical shift of the CYCH in a helical polypeptide is thc same in pure chloroforni as in the presence of trifluoroacetic acid, while in the random coil conformation (in chloroform/trifluoroacetic

NMR SPECTROSCOPY

OF AMINO ACIDS, PEPTIDES, AND PROTEINS

483

acid) the chemical shift of the aCH is exactly that predicted from studies of oligopeptides in D20 (Nakamura and Jardetzky, 1967, 1968) (see Section 111). Any contribution of solvent effects to the change in chemical shift during the helix-coil transition must therefore be minimal. In general, the changes in chemical shift of the aCH peak with solvent composition in the transition region correlate closely with changes in optical rotatory dispersion (ORD) (specifically with the parameter bo; Moffitt and Yang, 1956). Bradbury et al. (1968b) have reported one apparent exception to this: a change in the pH of a n aqueous solution of copoly (L-glutamic acid42,~ - l y s i n e ~-alanine~O) ~~, produced changes in bo suggesting the formation of a maximum of SO% helix; however there was no change in either the position or the line width of the a C H peak. There is some doubt about the reliability of ORD measurements as a n indication of helix content in polymers of this type; since a t all pH values either the glutamic acid or the lysine side chains, or both, would be charged, it is unlikely that this copolymer ever forms a true a-helix. This is reflected in the absence of changes in its N MR spectrum. The change in the chemical shift of the aCH peak, unlike ORD measurements (Adler et al., 1968; Vournalcis et al., 1968), is quite insensitive to the nature of the side chain, and probably gives a more generally reliable measurement of the helix content of a polypeptide. 2. On helix formation the NH resonance shifts downfield by about 0.2 ppm. I n a right-handed helix of an L-amino acid, the NH proton will be immediately above the plane of the preceding peptide bond. This n-ould tend to shift the NH resonance upfield. On the other hand, participation of this proton in a hydrogen bond will tend to cause a shift downfield. In the majority of polypeptides, therefore, the effect of hydrogen-bond formation is dominant. The helix of poly-0-methylaspartate is a n exception, in that the NH shifts upfield. This is consistent with the suggestion made on the basis of ORD measurements (Karlson et al., 1960) that the hydrogen bonds in this left-handed helix are weaker. Their contribution to the net chemical shift is therefore smaller. 3. The NH and aCH resonances are markedly broadened on helix formation. Ferretti and Paolillo (1969) have reported an apparent exception to this generalization. In their spectra of poly-L-alanine (DP 40), double peaks were seen for the NH and for the aCH resonances, and were assigned to molecules in the helical and random coil conformations (see below). However, the peak assigned to the helical conformation was always sharper than that assigned to the random coil conforniation. Furthermore, determinations of percentage helicity based on the relative areas of those two peaks led to values which disagreed not only with the results of ORD (see Fasman, 1967), but also with earlier NMR studies on another sample of

484

G . C . K. ROBERTS AND OLEG JARDETZRY

poly-L-aianine DP 40 from the same batch (Markley et al., 1967), and no change in the relative areas of the two peaks was seen on raising the temperature 50" in 80% TFA in CDCI3 (Ferretti and Paolillo, 1969). The assignment of these two resonances t o the helix and random coil in this polymer must therefore be questioned, and t,he possibility of conformational impurities in the poly-L-alanine used (Hanlon, 1966) must be considered. The resonances of side chain protons show no change in chemical shift on helix formation; any such change in proteins must therefore be ascribed to tertiary rather than secondary structure. The extent of line broadening falls off with increasing distance from the backbone, reflecting increased motional freedom relative to the rigid helical backbone. Markley et al. (1967) and Stewart et al. (1967) observed single peaks for the CYCHand NH resonances throughout the helix-coil transition. However, Ferretti (1967; Ferretti and Paolillo, 1969) reported that a t intermediate helix contents double peaks were seen for both these resonances; this finding was confirmed by Bradbury et al. (1967a,b, 1968a) [the maximum in the line width of the aCH peak a t intermediate helix content seen by Markley et al. (1967; see Fig. 9) was possibly due to the existence of a n unresolved double peak]. Table V I summarizes the instances of single or double peaks for the aCH and NH resonances reported to date. Ferretti (1967; Ferretti and Paolillo, 1969) and Bradbury etal. (1967a,b, 1968a) concluded from this finding that the separate peaks represent helices and random coils respectively, that the rate of exchange of an individual residue between a helical and a random coil state falls into the slow exchange region of the NMR time-scale (see Section II,D), and thus that the exchange rate was less than about 10 sec-1. However, this interpretation is inconsistent with the results of chemical relaxation studies, which have indicated a relaxation time of 0.2-1.0 x 10-6 sec for the helix-coil transition by a variety of methods [temperature-jump ORD (Lumry et al., 1964), viscoelastic relaxation (Wada et al., 1967), ultrasonic absorption (Parker et al., 1968; Hammes and Roberts, 1969) ; dielectric relaxation (Schwarz and Seelig, 1968).I These two series of observations can be reconciled most simply by supposing that the existence of separate peaks reflects the molecular weight heterogeneity of the polymer samples rather than slow exchange. The exact solvent composition a t which the helix-coil transition takes place is known to depend on the chain length for short polypeptides (see Fasman, 1967). Thus, if a given polypeptide sample contains niolecules with a distribution of chain lengths about the nominal value, the helix-coil transition for this sample will appear to take place over a range of solvent composition, the actual transition of a particular molecule being much sharper than the apparent overall transition. At any given solvent composition within the

PEPTIDES, AND PROTEINS

N M R SPECTROSCOPY OF AMINO ACIDS,

TABLE VI Single and Double Peaks Reported

-~

Polypeptide Poly-halanine

Poly-D-alanine Poly-D-a-amino-nbutyric acid Poly-p-benz yl-Laspartate Poly-y-benzyl-Lglutamate

Poly-L-glutamate Poly-L-leucine

DP' 40 40 280 2 300 700 ? ?

760 ? ? 13 21 92 340 640

Double peak Single peak

+ +(?)

+

+ + +

(+)

+ + + + + + + + + +

? ? ? 800 Not observed due to Poly-~-lysine broadening Poly-L-methionine 280 ? Poly-D-norleucine Poly-kphenylalanine ? Copoly(~-Glu~~,~Lys~*, L-AI~~O)

+

+ +

a

DP

485

=

+ +

Reference Ferretti and Paolillo (1969) Markley et al. (1967) Markley et al. (1967) Ferretti and Paolillo (1969) Stewart et al. (1967) Bradbury et al. (19674 Bradbury et al. (1967a) Markley d al. (1967) Ferretti (1967) Marlborough et al. (1965) Bradbury et al. (196%) Bradbury et al. (1968a) Bradbury et al. (1968a) Markley et al. (1967) Bradbury et al. (1968a) Markley et al. (1967) Bradbury et al. (1968b) Markley et al. (1967) Stewart et al. (1967) Ferretti (1967) Bradbury et al. (1967a) Bradbury et al. (196813) Markley et al. (1967) Bradburv et al. (1967a) Conti and Liquori (1968) Bradbury et al. (196813)

degree of polymerization = average chain length.

transition range, some molecules will be predominantly helical and others predominantly in the random coil state, depending on their molecular weights. Since the transition for a given molecule is very sharp, the proportion of molecules with intermediate helix content will be very small. Thus, one will observe two separate peaks, representing two separate populations of polymer molecules: one corresponding t o the population mostly in helical form, with a chemical shift approximating that of the helix, and the other corresponding to the population mostly in random coil form, with a chemical shift approximating that of the random coil, b u t both rapidly exchanging between helix and coil. This interpretation is the

486

G. C. K . ROBERTS AND OLEG JARDETZKY

only one consistent with the relatively narrow line width of the two peaks. In the absence of rapid exchange, the width of the peaks from the rigid helical rod would be several orders of magnitude greater. Since the chain-length dependence of the transition point disappears for long chains (Schwarz, 1967; Applequist, 1968), this model predicts that high molecular weight saniples (DP 100-200) would show only a single peak. Such a molecular weight dependence of the appearance of the spectrum has been demonstrated by Bradbury et al. (1968a) with poly-y-benzyl-Lglutamate. They observed separate peaks for samples of degree of polymerization (DP) 13, 21, and 92, but a single peak for a sample of DP 640. Ferretti and Paolillo (1969) have stated that double peaks for the N H resonance can be seen in a poly-L-alanine sample of DP 2 300. Their spectra, taken a t 220 MHz, show a distinct shoulder o n the upfield side of the NH peak (whose position is that corresponding to the helical conformation). This shoulder is, however, only 0.15 ppm upfield from the main peak, while the difference in chemical shift between the NH peak in the helical and random coil forms is 0.35 ppm for poly-L-alanine DP 40 (Ferretti and Paolillo, 1969). It is therefore doubtful that the shoulder corresponds to the random coil conformation. A definitive test of the idea that molecular weight heterogeneity is responsible for the double-peak phenomenon will be possible when completely monodisperse polypeptide samples become a ~ a i l a b l e . ~ I n this context it is also interesting to note that in the spectra of short polypeptides, particularly in the helical conformation, small peaks can be seen in the aCH region which can be assigned to residues a t the end of the chains. For poly-y-benzyl-L-glutamate of DP 13 and DP 21, Bradbury et al. (1968a) calculated that two residues a t each end of the chain remained nonhelical.

V. NMR

OF

PROTEINS

There are two general types of problem in protein chemistry that can in principle be solved by high-resolution NMR. The first embraces problems of the three-dimensional structure of proteins on solution. The second is the structure of binding sites and the mode of bonding of small molecules. Perhaps the most noteworthy feature of NMR as a structural method is that it allows one to define the structure of a protein in solution in considerable detail and to follow structural changes as functions of time. Three problems of protein structure fall into the first category: Note added in proof: E. M. Bradbury (personal communication) has recently subjected this interpretation to an experimental test and reports it to be borne out by his findings.

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

487

1. The complete definition of the conformation of a protein in solution.

It should be noted that this is in principle possible on the basis of high resolution NMR data alone. However it is an extremely difficult and

laborious task and has thus far not been attempted. 2. The mechanisms of folding arid unfolding of protein chains. Little detailed work on this question has been done. However, the high information content of a protein NMR spectrum should allow one to distinguish between a two-step and a multistep mechanism of denaturation and to describe precisely the sequence of structural changes. 3. Changes in conformation involving individual amino acid residues or entire regions of the polypeptide chain. Both conformational equilibria in the protein itself and conformational changes produced by ligand binding have been detected by NMR and will be discussed below. I n addition to providing estimates of the rates of such changes, NMR makes it possible t o define the amino acid residues involved. The majority of the successful investigations of protein by high resolu-

NO OF PROTONS IN EACH PEAK 800

I5

12

3

6 \3 I

12

750

3

3 I

700

c p s from HMS

FIG.10. Aromatic region of the NMR spectrum (100 MHz; time-averaged over 25 scans) of hen egg-white lysozyme in 8 A4 urea containing excess 2-mercaptoethanol a t 65°C. Protein concentration 0.0068 M . The assignments are shown by the lines below the spectrum, which represent the spect,ra of the amino acids tryptophan (-), phenylalanine (---), and tyrosine (-.--) obtained under the same conditions. The number of protons in each peak was obtained by multiplying the number of protons contributing to that peak in the amino acid by the number of moles of each amino acid per mole of enzyme (Phe 3, Trp 6, Tyr 3). From Cohen and Jardetzky (1968).

488

G. C. K. ROBERTS AND OLEG JARDETZKY

tion NMR to date have been directed to the solution of the second group of problems, i.e., the binding of small molecules to proteins. The emphasis on such problems in the present review simply reflects this fact. The first NMR spectrum of a protein, ribonuclease, was reported by Saunders et al. (1957). The four broad peaks obtained a t 40 MHz were accounted for by Jardetzky and Jardetzky (1957) in terms of the spectra of the constituent amino acids. The assignments were of course very crude and the fit was approximate; subsequent work (Cohen and Jardetzky, 1968; McDonald and Phillips, 1969a) has clearly shown that it is in fact the spectrum of the fully denatured protein that can be accurately accounted for by this procedure (see Fig. 10). Bovey and Tiers (1959), Saunders and Wishnia (1958), Wishnia and Saunders (1962), Kowalsky (1962, 1964), and Mandel (1964, 1965) have collected NMR spectra of several proteins (ribonuclease, lysozyme, bovine serum albumin, cytochrome c, insulin, aldolase, myoglobin, chymotrypsin, pepsin) a t 60 and 100 MHz. I n all these cases, only broad, largely unresolved envelopes were observed. This is due to both the extensive overlap of the amino acid spectra (Section 11) and the relatively large widths of the individual lines. Since the line widths are proportional to the inverse of the rate of molecular tumbling and hence to the molecular weight, they tend to be larger in the larger proteins. Although a strict correlation between line widths and molecular weight does not hold because of the possibilities of local motional freedom, in general the effective resolution is less in the case of larger proteins. Thus far (with few exceptions) useful spectra have been obtained only for proteins in the molecular weight range below 25,000. Resolution is understandably of major importance in protein NMR spectroscopy since the amount of information t h a t can be obtained from a spectrum is directly proportional to the number of lines which can be individually resolved. It had been hoped that the use of high field strength spectrometers would provide a general solution to this problem; however, protein spectra a t 220 MHz show only a marginal improvement over those obtained a t 100 MHz. It can easily be calculated from the chemical shifts of the amino acids that even a t 1000 MHz the resolution of a protein spectrum would be far from complete. The main advantage of the high field strength spectrometers lies in their higher sensitivity. The ultimate solution of the resolution problem lies in selective isotopic substitution (Jardetzky, 1965; Markley et al., 1968a).

A . Assignment of Resonances To obtain specific information from the spectrum it is first necessary t o assign each individual resonance not only to an amino acid entity, but to a

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

489

specific residue in the protein. The obvious prerequisite for such a complete assignment is that the amino acid sequence of the protein be known. I n an ordinary native protein the only resonances that can be resolved, and therefore assigned unequivocally to a particular amino acid, are those on the outer edges of the spectral envelope: the C2-H resonance of histidine (Bradbury and Scheraga, 1966; Meadows et aZ., 1967; Bradbury and Wilairat, 1967) and the N H resonance of tryptophan (McDonald and Phillips, 1968; observed only in HzO solution). The identification of the histidine C2-H resonance is particularly unambiguous, since titration curves can be obtained for the imidazole group by following the change in chemical shift of the resonance with p H (see Fig. 11). I n addition, peaks shifted t o unusually high field (up t o 1.0 ppm up$eZd from DSS) are observed in some proteins, such as lysozyme, and have been attributed to methyl or methylene groups close to the faces of aromatic RNase -HISTIDINE TITRATION CURVES

a 0 0

C4 -HIS

PH

FIG.11. Titration curves of the imidazole groups of the four histidine residues of bovine pancreatic ribonuclease. The chemical shift (in Hz a t 100 MHz) of the imidazole C2-H and C4-H resonances are shown as a function of pH (meter reading, in 0.2 M sodium acetate in D20). From Meadows et al. (1968).

490

G. C. K. ROBERTS AND OLEG JARDETZKY

rings (McDonald and Phillips, 1967; Sternlicht and Wilson, 1967). However, shielding by aromatic rings is a considerably more tenuous argument for assignment, since it is in this case impossible even to guess the nature of the amino acid residue without knowledge of the crystal structure. Perturbation of resonances by the binding of small molecules can sometimes be used for assignment on the basis of additional external information (McDonald and Phillips, 1969b), but onZy provided the observed spectral changes are not also used to derive further information on the nature of the binding process. Otherwise the argument clearly becomes circular. Proteins containing heme prosthetic groups show resonances far to highand low-field of the normal range (Kowalsky, 1965; McDonald and Phillips, 1967; Wuthrich et al., 1968a,b; Kurland et aZ., 1968; Davis et al., 1968, 1969) (Fig. 12). These resonances can be assigned t o (a) protons of the porphyrin ring itself and (b) protons of the protein close to the prophyrin ring. I n

n b I I I

t3

+1

+5ppm

C

-25

I

I

-20

- I5

_ .

-1Opprn

M FIG.12. NMR spectra (220 MHz) of sperm whale cyanometmyoglobin, 5 X in deuterated phosphate buffer, pD 6.6, at 35°C. Chemical shifts are given as parts per million (downfield negative) from 2,2-dimethylsilapentane-.i-sulfor~ic acid (DSS). (a) Range +1.5 to -10 ppm. The five sharp lines betwecn -3.5 and - 6 ppm correspond to the residual water resonance and its first atid second spinning side bands. (b) Range 0 to +5 ppm (high field). (c) Range -10 to -30 ppm (low field). From Wuttrich et at. (1968a).

NMR SPECTROSCOPY

OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

491

both these cases, the large shift is due to the considerable ring-current anistropy of the porphyrin ring and/or to contact or pseudo-contact interactions (see Section II1,C) with the paramagnetic iron atom of the heme. An almost complete assignment of these resonances in myoglobin and hemoglobin has been made, and detailed conclusions about the distribution of electron density in the heme have been drawn from the magnitude of the contact shifts (Wiithrich and Shulman, 1968, 1969). As already pointed out, the only general method by which peaks in the main body of the spectrum can be assigned is selective deuteration of the protein chain (Jardetzky, 1965) [although curve-fitting (Cohen and

x2 n

FIG.13. Comparison of a portion of the aliphatic region of the NMR spectra of staphylococcal nuclease (Nase), a selectively deuterated analog (Nase-D5) and the 'H-laheled amino acids present in Nase-Dj. The doithlet X2 is a dialyzable impurity. (a) Kase, pH 7.25, 50 scans; (b) Nase-DS, 100 scans; (c) peak positions of 'H-labeled amino acids present in Nase-DJ. From Markley (1969).

492

G. C. K. ROBERTS AND OLEG JARDETZKY

Jardetzky, 1968; Markley, 1969) has a certain limited value for the analysis of the relatively simple aromatic region]. The method requires the synthesis of a protein in which all the amino acids except two or three are deuterated; the proton NMR spectrum will then consist only of the resonances of these two or three amino acids. The biosynthesis of such selectively deuterated analogs has recently been achieved in two laboratories (Markley et al., 1968a, 1969; Putter et al., 1969a;b; Crespi et al., 1968). Figures 13 and 14 show a comparison of the aromatic and aliphatic regions of the NMR spectra of normal and selectively deuterated staphylococcal nuclease (Markley et al., 1968a). The difference is quite dramatic, and it is clear that by the preparation of a series of such selectively deuterated analogs the entire NMR spectrum can be analyzed in terms of amino acid entities. The further problem of assigning each peak to a particular residue in the amino acid sequence is clearly more formidable. Assuming that adequate resolution can be obtained [even in a selectively deuterated enzyme, the

FIG.14. Comparison of the aromatic region of the NMR spectra of staphylococcal nuclease (Nase) and a selectively deiiterated analog (Nase-D4). Assignments: His, H1, H2a, H2b, H3, and H4 (the low-field peaks are C2 and the high-field peaks C4 imidazole protons); Tyr, Y1-7; Trp, W (C-2 ringproton). (a) Nase, pH 6.0,75sctms, (b) Nase-D4, pH 6.0, 228 scans. From Markley (1969).

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

493

resonances of all the tyrosine residues, for example, are not separately resolved (see Fig. 14)], there are three approaches that have been adopted to make individual assignments. 1. Minor chemical modification of a single residue should, ideally, perturb only the resonance of that particular residue. This, however, is not always the case. It is well known that chemical modification of a protein always carries with it the danger of producing more or less subtle conformational changes. Furthermore, the modifying group may affect the shielding of neighboring residues directly. A complete and unambiguous assignment may therefore not be possible, but a partial assignment can often be achieved and an incorrect assignmerit is unlikely. Assignments have been made in this way for ribonuclease (Meadows et al., 1968), lysozyme (McDonald and Phillips, 1968), and staphylococcal nuclease (Putter et al., 1969a,b; Markley et al., 1969). 2. The use of ((mutant” proteins which differ by known amino acid substitutions is for this purpose equivalent to chemical modification. Since the proteins are still functional, major conformational changes cannot have occurred, though minor changes cannot be ruled out. This approach has been used for cytochrome G by Glickson et al. (1968), and for staphylococcal nuclease by Markley, Williams, and Jardetzky (see Markley, 1969). I n the case of the staphylococcal nuclease, the enzyme was isolated from the usual Foggi strain of Staphylococcus aureus, and also from the V8 strain, in which a single substitution of leucine for histidine has occurred at position 124 in the Foggj nuclease. Figure 15 shows the histidine C2-H region of the spectra of the two enzymes; it is clear that one peak is present in the enzyme from the Foggi strain, but not in that from the V8 strain, and titration curves establish unequivocally that it is peak 3 t h a t is absent. Histidine peak 3 can therefore be assigned to histidine 124 in the Foggi nuclease. 3. I n those cases where one or two peptide linkages in a protein can be cleaved by a proteolytic enzyme and the fragments recombined to give a functional entity, specifically deuterated “hybrid” proteins can be prepared. Thus, if an enzyme is cleaved into two fragments which on mixing regenerate an active enzyme, then one fragment can be deuterated (by exchange, biosynthesis, or chemical synthesis) and recombined with a normal hydrogen-containing fragment. Now only those residues in the nondeuterated part of the molecule will appear in the NMR spectrum. This method has been used for ribonuclease (Meadows et al., 1968) and for staphylococcal nuclease (Markley, Williams, and Jardetzky, unpublished experiments). To give a clearer idea of the procedures involved, the assignment of the four histidine C2-H resonances of ribonuclease (Meadows et al., 1968) will be discussed in more detail; this was the first case in which a single resonance

494

G. C. K . ROBERTS AND OLEG JARDETZKY

( 0 )

(C)

(d)

9.0

8.5

,s

8.0

7.5

7.0

(PPm)

FIG.15. Comparison of the aromatic region of the NMlt spectra of staphylococcal nuclease (Nase) from the Foggi and V8 strains of Staphylococcus uureus. (a) Nase Foggi, pH 6.00, 75 scans. (h) Nase V8, pH 6.00, 64 scans. (c) Nase Foggi, pH 7.25, 75 scans. (d) Nase V8, pH 7.25, 58 scans.

in a protein NMR spectrum was assigned unequivocally to a particular residue in the protein. The approach used initially was that of chemical modification; iodoacetate reacts with ribonuclease to form two alkylated derivatives, 1-carboxymethy I-histidine 119-ribonuclease and 3-carboxymethyl-histidine 12-ribonuclease (Crestfield et al., 1963; no dialkylated derivative is formed). The titration curves of the histidine residues of these derivatives, obtained from their NMR spectra, are shown in Figs. 16 arid 17. In both cases the alkylatiori of a single histidine residue produces large changes in the pK value of two histidines, indicating either that tt conformational change has occurred, or that two histidines are close together in the three-dimensional structure of the enzyme. Since the same two histidine peaks are affected by alkylation in both positions, the latter explanation is the more likely. I t is consistent with the inferences drawn from chemical modification studies (Crestfield r t al., 1963), and r+\iththe crystal structure of the enzyme (Kartha et aE., 1967; Wyckoff et al., 1967). The chemical modification experiments thus allow the histidine pealis t o be divided into two groups; peaks 2 and 3 (numbering as in Fig. 11) corre-

K M R SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

-- .

9oc

495

-RNASE A - - - - 3 - CM - H i s - 12-RNAsE

86C

8I E

2

c

g u

82C

78C

74c I

I

I

I

5.0

6.0

7.0

8.0

PH

Fig. 16. Titration curves of the histidine residues of ribonuclease A (-) and 3-carboxymet>hyl-histidine 12-ribonuclease (- - -). The ordinate is the chemical shift of the C2-H or C4-H peaks, in Hz (at 100 MHz) downfield from hexamethyldisiloxane (HMS). From Meadows et al. (1968).

sporid to histidines 12 and 119, and peaks 1 and 4 to histidines 48 and 105. In order to obtain a complete assignment, a much more subtle modification is required, so as to affect the C2-H resonance of either histidine 12 or histidine 119 but not both. The obvious alternative is specific deuterium substitution, and this was accomplished by using the modified enzyme ribonuclease S. This enzyme is identical t o ribonuclease A except for cleavage of the peptide bond between residues 20 and 21, and retains full enzymatic activity (Richards arid Vithayathil, 1959). The histidine titration curves of ribonuclease A and ribonuclease S are compared in Fig. 18. Peak 1 is quite unaffected, peaks 2 and 3 show a slight increase in pK (by about 0.5 pH unit) but no change in chemical shift in either the protonated or the unprotonated form. Peak 4, on the other hand, is shifted 20-30 Hz downfield in riboriuclease S as compared to ribonuclease A. On the basis of the abnormal chemical shift of this peak a t low pH in ribonuclease A, it was suggested that it could be assigned to histidine 48, the “buried”

496

G . C. K . ROBERTS AND OLEG JARDETZKY

G His C,

5.0

7.0

6.0

8.0

Ph

FIG.17. Titration curves of the histidine residues of ribonuclease A (-) and l-carboxymethyl-histidine 119-ribonuclease (- -). The ordinate is the chemical shift of the C2-H or C4-H peaks, in Ha (at 100 MHz) downfield from hexamethyldisiloxane (HMS). From Meadows et al. (1968).

-

histidine residue of ribonuclease A (Kartha et al., 1967). The partial normalization of the chemical shift of this peak in riboriuclease S is further evidence for this assignment, since histidine 48 is in the vicinity of the peptide bond between residues 20 and 21 (Kartha et al., 1967) and becomes less (‘buried” when this bond is cleaved (Wyckoff et al., 1967). Ribonuclease S was then separated into its two components, S-peptide (residues 1-20) and S-protein (residues 21-124), and the imidazole C2 hydrogen of histidine 12 was exchanged for deuterium by incubating the S-peptide in D20 (at pH 7) for 5 days a t 40”;after this time the C2-H resonance was unobservable. The two components were next recombined to give ribonuclease S’, whose NMR spectrum contained only three histidine peaks (Fig. 19). A comparison of the titratiori curves of ribonuclease S and ribonuclease S’ (Fig. 20) indicated that either peak 1 or peak 2 was absent

N M R SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

497

900

860 #

I

I

E 2

. I -

820

; 1087 ul

(1

0

740

5.0

6.0

7.0

8.0

PH

FIG. 18. Titration curves of the histidine residues of ribonuclease A (-) and ribonuclease S (- - - -). The ordinate is the chemical shift of the C2-H or C4-H peaks, in Hz (at 100 MHz) downfield from hexamethyldisiloxane (HMS). From Meadows et al. (1968).

-

in ribonuclease S'. Since the chemical modification experiments had already shown that peak 1 was not histidine 12, the exchanged proton must be that of peak 2 . A complete and unambiguous assignment can thus be made: Peak

pK (32", 0.2 M sodium acetate)

Histidine residue

6.7 6.2 5.8 6.4

105

12 119 48

The most immediate result of the assignment of these histidine peaks is that, for the first time, the pK values of individual residues in a protein have been determined (see Roberts et al., 1969~). Furthermore, it made possible a

498

G. C. K. ROBERTS AND OLEG JARDETZKY

detailed interpretation of the NMR studies of inhibitor binding to ribonuclease (see Section V,C). I n summary, selective deuteration is a wholly general method for the assignment of resonances in a protein NMR spectrum to particular amino acids. The correspondingly general method for assigning resonances to individual residues is chemical synthesis of proteins using deuterated amino acids. With the recent synthesis of ribonuclease (Gutte and Merrifield, 1969; Denkewalter et al., 1969, and subsequent papers), it is in principle possible to make a complete assignment of the entire NMR spectrum of a protein by the synthesis of a sufficient number of specifically deuterated analogs. Short of a massive undertaking such as this, the most promising approach seems to be the preparation of “hybrid” specifically deuterated proteins, as described above for ribonuclease.

h

FIG.19. Comparison of the imidazole C2-H region of the NMIt spectrrim of riboIiuclease S and ribonuclease 8’ (in which the C2-H of histidine 12 has been exchanged for deuterium). The curves below the spectra were obtained with a duPorit curve resolver, and show the decomposition of each spectnim into three or four Lorentziari peaks of equal area. From Meadows et al. (1968).

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

RIBONUCLEASE S’

I 900

499

--*---RNASE s‘

-

-

RNASE S

v,

860-

E

-

?

-4-

In a

u

820

-

cI 1

1

5.0

I

I

6.0

I

I

7.0

I

I

8.0

I

PH

and FIG.20. Titration curves of the histidine residues of ribonuclease S (-) ribonuclease S’, in which the C2-H of histidine 12 has been exchanged for deuterium (- - - - -). The ordinate is the chemical shift of the C2-H peaks, in Hs (at 100 MHs) downfield from hexamethyldisiloxane (HMS). From Meadows et al. (1968).

B . The Binding of Small Molecules to Proteins The problem of the structure of binding sites-in its various forms, such as inhibitor-enzyme, hapten-antibody or drug-receptor interactions-is particularly well suited to analysis by high resolution NMR. The conceptual basis for such an analysis is simple and is shown diagrammatically in Fig. 21. Let us assume that we have a small molecule consisting of three chemical groups, which we will designate A, B, and C, and a corresponding protein site, consisting of groups (parts of amino acid residues), which we will designate D, E, F, G, and H. For each of these groups A-H there will be a corresponding absorption line on the NMR spectrum, whose characteristics in the absence of binding can usually be measured. When the small molecule binds to the protein site, some groups will be in direct contact with each other (Fig. 21). A priori the number of possible combinations of contacts is very large, given by the formula N = n(2m- 1) (n - l)(am- 1)2 (n - 2)(2m. . . 1(2m- l)n, where n is the number of groups on the small molecule and m the number of groups in the binding site of the

+

+

+

500

G. C. K. ROBERTS AND OLEG JARDETZKY

FIG. 21. Schematic representation of the binding of a small molecule, with functional groups A, B, and C, to 8 site on a macromolecule containing functional groups D, E, F, G, and H. protein. I n the case illustrated n = 3, m = 5, and N = 31,806. The problem is thus to determine which of these possible combinations actually occurs. One might expect that the most pronounced spectral changeswhether affecting chemical shift or line width-will be found in lines originating from groups interacting directly, as shown in Fig. 21, and that it should therefore be possible to infer the structure of the complex from the differential changes in the NMR spectrum. I n pursuing this line of argument in a particular case, it is of course necessary to bear in mind that proximity is not the sole factor in determining the magnitude of the spectral changes; the magnitude and distribution of local charges, relative orientation of the different groups, group polarizabilities, and other factors entering into the calculation of intermolecular forces must be considered in detail. Such a detailed discussion is beyond the scope of this review (see

NMR SPECTROSCOPY OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

5081

Jardetzky, 1964; Emsley et al., 1965). The simplified argument is given merely to outline the general procedure for mapping the structure of the complex on the basis of differential changes in the high-resolution NMR spectra. The prototype of this kind of experiment was reported by 0. Jardetzky, J. J . Fischer, and P. Pappas (1961; Fischer and Jardetzky, 1965). I n the last six years several similar NMR studies have been published. For purposes of discussion they can be divided into two groups: (1) those in which only the spectrum of the ligand was observed, identifying the interacting groups on the small molecule, and (2) those in which the spectrum of the protein was also studied (yielding a complete structure of the complex). 1. Studies of Ligand Spectra

As already indicated, when a small molecule binds to a protein, two types of change are commonly observed in its NMR spectrum: a change in chemical shift of one or more resonances, reflecting a change in the magnetic environment of one or more of the groups on binding, and/or a selective broadening (increase in relaxation rate) of one or more peaks, which can be attributed to decreased motional freedom of a particular part of the small molecule. Those groups that interact directly with groups on the protein would be expected to show both the largest change in magnetic environment and the largest decrease in motional freedom. Depending on the lifetime of the small molecule-protein complex, the appearance of the spectrum falls into one of three categories, referred t o as the fast, intermediate, and slow exchange cases, respectively (Table VII) . This distinction applies t o both chemical shift and line width changes and has been discussed in Sections II,D and II1,D. Many studies of inter- and intramolecular rate processes utilizing chemical shift differences between two states have been reported (see Jonas and Gutowsky, 1968; Binsch, 1968). The theory describing the appearance of the spectrum in the cases in which the two states differ only in relaxation time, but not in chemical shift, has been discussed by Zimnierman and Brittin (1957), Fischer (1964), Jardetzky (1964), and Fischer and Jardetzky (1965). It is important to note that the slow, intermediate, and fast exchange regions defined by the chemical shift and relaxation rate differences, respectively, correspond to different actual exchange rates (cf. Section 11,D). Since the clitli,ge in chemical shift on binding is unlikely to exceed 100 Hz, while the relaxation rate of a proton on the small molecule may increase by two to three orders of magnitude, the relaxation method is more sensitive to weak binding. The direction and magnitude of the chemical shift does, however, provide direct indication of the magnetic environment of the protons of the ligand when bound to the protein.

TABLEVII Chemical Shift and Line Width Changes Relative to Exchange Rate ~~

Exchange rate region Slow Intermediate

Fast

Chemical shift Definition

Appearance of spectrum

1

Separate narrow resonances for

1

each state Broad single resonance

r

N

27rAv

1 - > ZTAV

7

Relaxation time Definition 1

1

r

Tm

-<-<-

cal shift weighted average of two states

1

T~B

Narrow line superimposed on broad line

Tm

Line width determined by exchange rate, and therefore the same for all groups undergoing exchange

1 1 1 <-
Single line, line width weighted average of two states

1 1 1 <-<-

TZF

Single narrow resonance, shemi-

Appearance of spectrum

r

0 F

m

0

N M R SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

503

a. Binding of Xulfonamides to Serum Albumin. The work of Jardetzky and Wade-Jardetzky (1965) on the binding of sulfonamides to bovine serum albumin will serve as an example of the information that can be obtained from relaxation rate measurements. The spectrum of sulfacetamide

consists of a single peak a t high field due to the methyl group, and a n A2B2 quartet a t low field due to the aromatic protons. Figure 22 shows the effect of bovine serum albumin on the relaxation rates of these two groups of protons; the relaxation rate of the aromatic protons increases by a factor of 44, and that of the methyl protons by a factor of 14, as the protein concentration is increased from 0 to 10%. This selective broadening of the aromatic resonances must be due to a specific interaction with the protein. Nonspecific broadening mechanisms, such as an increase in viscosity, would be expected to affect all peaks to the same extent. This is in fact seen if a 18 16 -

14 -

I

3

5 BSA

7 10 CONCENTRATION

15 (Oh)

FIG.22. Relaxat.ion rates of 0.1 111 siilfacetamide peaks as a function of bovine serum albumin (BSA) concentration in DzO, pH 8.8. 0 , p-Aminobenzene sulfonamide (PABS) protons; A, methyl protons. From Jardetzky and Wade-Jardetzky (1965).

504

G. C. K. ROBERTS AND OLEG JARDETZKY

18. 16I4 -

12. 10-IP

8-

I,

-It--

6-

4. 21 2 3 4 5

10

16

dl FIG.23. Relaxation rates of sulfacetamide peaks as a function of the reciprocal of

the sulfaeetamide concentration ([S])at a constant concentration (10%)of bovine serum albumin, in DzO, pH 7.8. 0 , p-Aminobenaene sulfonamide (PABS) protons; A, methyl protons. From Jardetzky and Wade-Jardeteky (1965).

protein, such as y-globulin, which does not bind sulfonamides is used: the increase in relative line width is the same for all peaks and is proportional to the increase in viscosity. Furthermore, the relaxation rate of the sulfonaniide protons depends on the sulfonamide :albumin ratio rather than on the albumin concentration and, for a given albumin concentration, decreases with increasing sulfonamide concentration (Fig. 23). This is exactly the opposite to what one would expect if the broadening were due to 11 nonspecific mechanism. In the fast exchange limit, which judging from the appearance of the spectrum applies in this case (if the line widths were determined solely by the exchange rate, all lines would be equally affected)

whcre a is the fraction bound. Using a simple mass action model with n noniriteractirig binding sites on each albumin molecule, Ku

=

S(1 - a ) ( n A - as) CtA

(22)

NMR SPECTROSCOPY OF AMINO ACIDS,

PEPTIDES, AND PROTEINS

505

where S and A are the total sulfonamide and albumin concentrations, respectively, and K D is the dissociation constant of the complex. This can be rearranged t o give

If a << 1 (since the ligand is in large excess) and n

=

1, this simplifies to

1

S = -KD+-A LY

(24)

A plot of all sulfonamide concentrations that give some particular, arbitrarily chosen, line width against the corresponding albumin concentration will give a straight line of slope 1/a and intercept -KD, since lines of identical width correspond to identical values of a ; such a secondary plot M ; the is shown in Fig. 24. The value of K D obtained is 2 to 9 X 3.0 sec-'

I

2 3

4

5

6 7 8 9 1011 1213 1415 BSA CONCENTRATION (x)

FIG.24. Sulfacetnmide concentration versiis bovine serum albumin concentration for lines of constant width (l/Tt = 3.0 sec-l and 6.0 sec-l), pH 8.8. 0, p-Aminobenzene sulfonamide (PABS) protons; A, methyl protons. From Jardetzky and Wade-Jardetzky (1965).

506

G. C. K. ROBERTS AND OLEG JARDETZKY

TABLEVIII

Calculation of Relaxation Rates for Bound Sulfaeetamidea Averageb

PABSd PABS CH3 CHs

3 6 3 6

0.25 0.25 0.45 0.45

266 80 100 60

710 720 255 290

715

2870

275

610

* Jardetzky and Wade-Jardetzky (1965). b

I n sec-1. = fraction bound. PABS = p-aminobenzene sulfonamide.

c 01

values of a can be used to calculate (1/T2) bound, arid the results are shown in Table VIII. The quantity of primary interest is T2,frPe/T2,bound,which shows that the p-aminobenzene sulfonamide (PABS) moiety is preferentially immobilized by interaction of sulfacetamide with the protein. The findings that the binding is enhanced by increasing ionic strength arid reduced, but not abolished, by protonation of the sulfonamide group suggest that the aromatic ring is involved in a van der Waals’ (permanent and induced dipolar) interaction with groups on the protein. In the series of sulfonamides, the most interesting case is that of sulfaphenazole

The effect of bovine serum albumin on the PABS, pyrazole, and pheriyl resonances of this molecule are shown in Fig. 25. It is clear that the PABS and phenyl protons are broadened almost equally, and the pyrazole protons are much less affected. Although both the PABS arid phenyl rings must, therefore, he binding to the protein, they cannot be binding simultaneously, or the molecule would essentially be binding as a rigid unit, and the pyrazole protons would be broadened to the same extent as the PABS and pherlyl protons. The simplest model consistent w-ith these data is that there are two nonequivalent binding sites, one for the PABS group, and orie for the phcriyl group, and that the sulfapheiiazole niolecule is bound to orie of these sites a t a time. This idea is supported by the finding that phenylpropariol competes specifically for the PABS site, causing selective narrowing of the

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

1

3

5

7

10

507

15

BSA C 0 NC ENT R AT I0 N

FIG.25. Relaxation rates of 0.15 M sulfaphenazole peaks, p H 9.9, as a function of bovine serum albumin concentration. 0 , p-Aminobenzene sulfonamide (PABS) protons; A, pyrazole protons; 0, phenyl protons. From Jardetzky and Wade-Jardeteky (1965).

PABS resonances and broadening of the phenyl resonance of sulfaphonazole. Thus the relaxation method makes it possible to distinguish between nonequivalent binding sites for a small molecule bound to a protein with a heterogeneous population of binding sites. It should be noted however, that the method provides less information if the small molecule binds as a rigid unit; for example, in the case of cytidine 3' monophosphate binding to ribonuclease, the degree of broadening of the pyrimidine and ribose proton resonances and of the phosphate 31Presonance is almost identical (Roberts, Cohen, Denney, and Jardetzky, unpublished). I n the case of the binding of penicillin G to bovine serum albumin, the primary site of interaction with the protein was shown to be the phenyl group; this was confirmed by the observation that phenoxyacetic acid and phenoxyacetamide competed with penicillin G for the binding site, but N-acetyl4-aminopenicillanic acid did not (Fischer and Jardetzky, 1965). In similar cxperimcnts Gerig (1968) studied the binding of tryptophan to a-chymotrypsin, and Joff e (1967) the binding of p-nitrophenyl-/3-D-galactoside to antigalactosyl antibody. Hollis (1967) investigated the binding of oxidized and reduced nicotinamide adenine dinucleotide (NAD and X'ADH) to yeast and liver alcohol dehydrogenases. In the case of the

508

G . C. K. ROBERTS AND OLEG JARDETZKY

yeast enzyme, the resonances of the adenine protons of both NAD and NADH were broadened more than those of the nicotinamide protons, suggesting a specific interaction between the adenine moiety and the enzyme. [This result is the opposite of that obtained by Jardetzky et al. (1963); in the latter case, however, the enzyme was partially denatured.] When liver alcohol dehydrogenase was added to a solution of NAD or NADH, the coenzyme spectrum was reduced in intensity but not appreciably broadened; it appears that this is a case of slow exchange (see Table VII). The methyl resonances of ethanol and acetaldehyde were not broadened on addition of enzyme alone. Although slow exchange could not be ruled out, it was concluded that these compounds did not bind to the enzyme in the absence of coenzyme. b. Binding of Choline Analogs to Antibody. The binding of a number of choline analogs and derivatives to antiphenoxycholine antibody has been examined by Rurgen et al. (1067) and Metcalfe et aE. (1968). They concluded that the primary binding interaction was with the triniethylamnioniuni group; if two bulky groups were attached to the quaternary nitrogen (as in N-benzylacetarnidophenyl choline ether), the smaller of the two appeared to be forced into the binding site and was strongly imrnobilized. Methacholine, however, appeared to bind as a rigid unit, suggesting that there was a second binding site, probably for the acetyl group. This second binding site was not available to the acetyl group in acetamidophenyl choline ether, which is separated from the trimethylammonium group by a phenyl ring. Evidence was also presented that the trimethylammonium group of these compounds did not bind in exactly the same manner as the tetraniethylammonium ion. I n the case of acetamidophenyl choline ether, an anomalous temperature dependence of the line widths was observed. The line width of the N-methyl resonance went through a maximum a t about 30")while that of the acetyl methyl resonance decreased monotonically with increasing temperature. Since the relaxation rate of the N-methyl protons is greater than that of the acetyl methyl protons when the molecule is bound to the protein, this anomaly can be explained by postulating that the N-methyl protons are in the intermediate exchange region [(1/T2)frCc> 1 / > ~ ( l / T Z ) b o u n d ; see Table VII] below 30". In this temperature range, the line width of the N-methyl resonance will then be determined, not by (l/T2)bo,,nd,but by the rate of exchange of acetaniidophenyl choline ether molecules betn-een the free and bound states. c . Aspartate Transcarbamylase. I n a similar study, Schmidt et al. (1969) observed that the line width of the inethylene proton resonance of succinate was increased on binding to the catalytic submit of aspartate transcarbaniylase. This broadening was increased in the presence of carbainyl phosphate or its analogs, and since the tenipernture-dependence of the succinate

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

509

line width indicated that it was dominated by exchange, Schmidt et al. (1969) concluded that carbamyl phosphate increased the lifetime of the succinate-exzyme complex. This is consistent with the equilibrium dialysis results of Changeux et al. (1968). Since, in the absence of paramagnetic species, the spin-spin relaxation time, Tz, is sensitive to exchange, whereas the spin-lattice relaxation time, TI, is not, the observation that l / T zwas five times as great as TI is additional evidence for the conclusion that the line width of the succinate protons is dominated by exchange. Schmidt et al. (1969) also studied the line broadening of the methylene or methyl protons of a number of carbamyl phosphate analogs on binding to the catalytic submit of aspartate transcarbamylase. They concluded that there was a two-point attachment of these compounds to the enzyme, through the phosphate and carbonyl groups, respectively, and also that compounds such as N,N-dimethyl carbamyl phosphate are too large t o fit the binding site. d . Lysozyme. The first observation of a change in chemical shift of the resonances of a small molecule on binding to a protein was made by Thomas (1966, 1967), who studied the binding of N-acetyl-D-glucosamine to lyso-

*H-+

FIG.26. NMR spectra (60 MHz) of the acetamido methyl protons of N-acetyl-Dglricosamine, 0.05 M in 0.1 M citrate, pH L 5 . (A) Alone; (B) in the presence of 0.003 M hen egg-white lysozyme. The resonance a t the far left (lowest field) is that of 0.5% acetone, which was used as an internal standard. From Raftery et al. (1968).

510

G. C. K. ROBERTS AND OLEG JARDETZKY

zyme. This, and subsequent work on the same system by Dahlquist and Raftery (1968a,b; Raftery el al., 196S), is a good illustration of the information that can be obtained by studies of chemical shifts. When N-acetylD-glucosamine binds to lysozyme, the single resonance due to the acetamide methyl protons is split into two, both of which are shifted upfield (Fig. 26). By examining a freshly prepared solution of the P-anomer of N-acetyl-Dglucosamine and by comparison with the a- and @-methylglycosides, it was shown that the two acetamido methyl peaks correspond to the two anomeric forms of N-acetyl-D-glucosamine when bound to the enzyme (in free solution, the chemical shift of the acetamido methyl resonance is identical in both forms). Initially, the a- and P-methyl glycosides were used t o investigate this difference, since the problem of mutarotation does not arise for these compounds. If there is a simple mass-action equilibrium binding of the inhibitor (S) to a single site on the enzyme (E) [E S ES], and if the exchange of the inhibitor between the two states is rapid on the chemical shift time scale (see Table VII), then

+

where Kd is the dissociation constant, 6 is the observed chemical shift change, A is the total chemical shift difference between the bound and the free forms (so that 6/A is the fraction of inhibitor bound), and Eo and So are the total concentrations of enzyme and inhibitor, respectively (Dahlquist and Raftery, 1968a). When 6/A << 1 (so that 132/A2 -+0) this equation simplified to So

=

A 6

Eo -

- Kd

- Eo

(26)

A plot of Soagainst 1/6 will therefore have a slope of AEo and an intercept a t 1/13 = 0 of - (Kd Eo);plots of this type for a- and @-methyl-N-acetylD-glucosamine binding to lysozynie are shown in Fig. 27. From these curves, the following values were obtained : for the a-methyl glycoside, Kd = 5.2 X lop2M , A = 0.55 ppm, and for the &methyl glycoside, Kd = 3.3 X lov2M , A = 0.54 ppm. In this case, therefore, the magnetic environment of the bound acetamide methyl group is identical for the tlvo compounds, suggesting that they bind in a very similar orientation; the difference between them is in their binding constants. A similar conclusion was arrived a t by Sykes (1969b), though the values of Kd and A which he obtained differ somewhat from those of Dahlquist and Raftery (1968a). Alternative methods of analyzing chemical shift data to obtain binding constants and bound chemical shifts are given by Nakano et al. (1967), Spotswood et al. (1967), and Sykes (1968, 1969a). Both the Q- and p-anomers of N-acetyl-D-glucosaniine competed with both

+

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

511

I N

0 X

0

m

FIG.27. The reciprocal of the chemical shift change of the acetamido methyl protons (1/6), as a function of total inhibitor concentration (So) on addition of various concen trations of methyl N-acetyl-@-D-glucosamine (8-Me-GlcAc) or methyl N-acetyl-a-Dglucosamine (a-Me-GlcAc) to a constant concentration (0.003 M ) of hen egg-white lysozyme. From Raftery et al. (1968).

the a- and 0-methyl glycosides for binding to lysozyme, suggesting that all four compounds occupy the same or overlapping sites on the enzyme. By following the changes in chemical shift on mutarotation of freshly dissolved N-acetyl-a- or 0-D-glucosamine in the presence of lysozyme, Dahlquist and Raftery (1968a) were able to obtain values for the bound chemical shifts and dissociation constants of both these compounds : for N-acetylM , A = 0.68 ppm; for N-acetyl-P-D-glua-D-glucosamine, Kd = 1.6 M , A = 0.51 ppm. It is interesting that both cosamine, Kd = 3.3 X the dissociation constant and the total chemical shift change of the acetamido methyl protons on binding are essentially identical for N-acetyl-0-Dglucosamirie arid methyl N-acetyl-0-D-glucosamine, while there is an appreciable difference in both parameters between the a-anomer arid its methyl glycoside. This suggests that in the a-form the C(1)-hydroxyl group can act as a hydrogen bond donor to some group on the enzyme, and that the formation of this bond slightly alters the orientation of the sugar so as to change the magnetic erivironnient of the acetamido methyl group (Dahlquist and Raftery, 1968a). I n the other three compounds, no such bond can be formed, and the magnetic environment of this group is the same; the higher dissociation constant of the a-methyl glycoside may be

512

G. C. K. ROBERTS AND OLEG JARDETZKY

due to steric hindrance by the bulky methyl group (Neuberger and Wilson, 1967). This interpretation is consistent with the crystallographic studies of Blake et al. (1967). By studying the pH-dependence of the binding, Dahlquist and Raftery (196813) were able to demonstrate the existence of two groups whose titration affects the binding of methyl N-acetyl-P-Dglucosaniine. A group with an apparent pK of 4.7 affects the change in chemical shift of the acetamido methyl protons without affecting the binding constant, while a group of apparent pK 6.1 affects both the chemical shift change and the binding constant. The change in chemical shift of the C(1)-methoxy group is not affected by the titration of either of these groups. Dahlquist and Raftery (1968b) suggested on the basis of X-ray (Blake et al., 1967) and other data, that the two groups on the enzyme were glutamic acid 35 (pK = 6.1) and aspartic acid 103 (pK = 4.7). Dahlquist and Raftcry (1969) and Raftery et al. (1969) have subsequently extended these NMR studies of lysozyme-inhibitor interactions to the dimer, trimer, and tetramer of N-acetyl-D-glucosamine, and t o other related inhibitors. e. a-Chymotrypsin. The changes in 19Fchemical shifts of various fluorinated phenylalanines on binding to a-chymotrypsin have been studied by several workers (Spotswood et al., 1967; Zeffren and Reavill, 1968; Sykes, 1968, 1969a). At pH 6.0, the I9F resonances of N-acetyl-D-mfluorophenylalanine, N-acetyl-D-p-fluorophenylalanine (Spotswood el al., 1967), arid N-trifluoroacetyl-D-phenylalanine (Zeff ren and Reavill, 1968) shift downfield on binding (total chemical shift changes of 1.50, 1.47, and 1.75 ppm, respectively). At pH 7.8, however, the 19Fresonance of N-trifluoroacetyl-~-phenylalanineshifts upfield, with a total shift of 0.43 ppm (Sykes, 1968, 1969a). This difference indicates a major change in the magnetic environment of the fluorine atoms of the bound inhibitor with pH; Sykes (1969a) suggested that the titration of the histidine residue a t the active site might be responsible for this change. Upfield shifts of 0.060.10 ppm of all the resonances of sodium dodecyl sulfate on binding t o phycocyanin have been recently reported by Rosenberg et al. (1969). f. Binding to Proteins Containing Paramagnetic Metal Ions. Particularly detailed information can be obtained from changes in the spectrum of the small molecule on binding to a protein containing a paramagnetic metal ion a t the binding site. Studies of this type have been reported for Mn(I1)carboxypeptidase (Navon et al., 1968), pyruvate carboxylase (Mildvan and Scrutton, 1967), and pyruvate kinase (Mildvan et al., 1967). Carboxypeptidase normally contains 1 atomlmole Zn(II), but this can be replaced by Mn(I1) to give an active enzyme. If the diamagnetic Zn(I1) enzyme is used as a control, the broadening of the resonance lines of the inhibitor on binding must be due t o the proximity of the paramagnetic metal ion (see Navon et al., 1968). Now if there is no change in chemical shift,

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

513

where T 2 p is the paramagnetic contribution to the relaxation time, f is the fraction of the inhibitor bound to the enzyme, T ~ is Mthe relaxation rate of the protons in the bound ligand, and T M is the lifetime of the enzyme , so (metal)-inhibitor complex. If the exchange is slow, T M >> T ~ Mand l/TZp= j/rM,and for fast exchange, 7 M << 2 ' 2 ~and i/Tzp = j/TPM. These two cases can be distinguished by the temperature dependence of the line width (see Cohn, 1967) : in the slow-exchange limit, the line width is determined by ~ / T M and will increase with increasing temperature; the opposite is true in the fast-exchange limit, when the line width is determined by 1/T2M. Navon et al. (1968) showed that methoxyacetic acid, a weak inhibitor of carboxypeptidase, fell into the fast-exchange region, so that values of 1/T2M could be obtained. Solomon (1955) and Bloembergen (1957a,b) have shown that

- =1 - D ( 1 4rc+ TPM 15

1

+

3Tc

)+

W2TC2

CT,

where D, the dipolar interaction between the paramagnetic metal ion (with magnetic moment p ) and the inhibitor nuclei (with gyromagnetic ratio 7 ) a t distance r, is given by D = ( p 2 y 2 ) / r 6C ; is the isotropic hyperfine interaction constant, w is the nuclear Larmor precession frequency, T,, is the correlation time for the anisotropic dipolar interaction, and r e is the correlation time for the isotropic hyperfine interaction. This relationship can give a t least an approximate value of r , the distance between the observed nucleus and the paramagnetic metal ion. For methoxyacetic acid and Mn(I1)-carboxypeptidase, Navon et al. (1968) found r = 4.3 A for the methylene protons and r = 4.7 8 for the methyl protons. These values indicate that the carboxylate group of this inhibitor probably binds directly to the metal ion. If the binding of the inhibitor is such that the fastexchange condition is not obeyed, upper limits to the distance, r, can stiIl be obtained [since T ~ M 5 ( T ~ M T M ) ] and in the slow-exchange limit T M , the reciprocal of the dissociation rate constant, can be determined.

+

2. Studies of Ligand and Protein Spectra

a. Bovine Pancreatic Ribonuclease. The first protein whose binding site was more completely analyzed, by studying the spectra of the protein as well as of the small molecule, was bovine pancreatic ribonuclease A (Meadows, 1968; Meadows and Jardetzky, 1968; Meadows et al., 1969). This enzyme has two histidine residues (numbers 12 and 119 in the amino acid sequence) a t the active site (Crestfield et al., 1963), and the C2-H resonances of these arid the other two histidines can be resolved; their assignment

514

G. C. K. ROBERTS AND OLEG JARDETZKY

(Meadows et al., 1968) has been discussed above. Figure 28 shows the changes produced in the histidine C2-H region of the NMR spectrum of ribonuclease on addition of the inhibitor cytidine %‘-monophosphate (3’-CMP) a t pH 5.5. The shifts are shown as a function of 3’-CMP concentration in Fig. 29; it can be seen that the C2-H peak of histidine 105 is unaffected throughout, while the peaks of histidine 48, 12, and 119 shift downfield by 7, 12, and 60 Hz, respectively (at 100 MHz). At pH 7.0, the shifts are qualititatively similar, but the peaks of both histidine 12 and histidine 119 shift much farther downfield. The pH dependence of the chemical shift changes on 3’-CMP binding can best be understood by reference to Fig. 30; this shows the titration curves of the histidine residues of ribonuclease in the presence and absence of 0.04 M 3’-CMP (at this concentration, the enzyme is saturated with inhibitor in the pH range 5-7). Histidine 105 is wholly unaffected in pK and chemical shift, while the peak of histidine 48 is shifted downfield 7 Hz a t low pH (above pH 5.5 in sodium A

C 12

%MP C,H

105 I2 12

950

3-CMP

4a

900 CHEMICAL

a50

105

800

SHIFT

FIG. 28. Imidazole C2-H and C4-H region of the NMIt spectrum (100 MHz) of ribonuclease A [0.0065 M in 0.2 M NaCl (DiO), p H (meter reading) 5.5, 32”C] in the presence of increasing concentrations of cytidine 3’-monophosphate (3’-CMP). Peak 105 a t 780 Hz is imidazole C4-H peak of His 105. Other peaks labeled 105, 12, 119, and 48 are imidazole C2-H peaks. (A) RNase alone, time-average of 25 scans; (B) +0.002 M 3’-CMP, 100 scans; (C) +0.005 M 3’-CMP, 39 scans; (D) $0.010 M 3’- CMP, 68 scans; (E) +0.030 M 3’-CMP, 100 scans. Chemical shift, scale is in units of Hz downfield from HMS. From Meadows and Jardetzky (1968).

NMR SPECTROSCOPY

OF AMINO ACIDS, PEPTIDES, AND PROTEINS

i

515

105

0.010

0.020

r 3‘- C M P1

0.030

0.040

FIG.29. Chemical shifts (in H a a t 100 MHz downfield from hexamethyldisiloxane) of ribonuclease A histidine C2-H and (at 780 Hz) C4-H peaks as a function of total added 3’-CMP concentration. Ribonuclease concentration 0.0065 M in 0.2 M NaCl (DsO), p H (meter reading) 5.5, 32°C. Data of Meadows and Jardetzky (1968).

chloride this peak broadens and becomes undetectable-see below, Section IV,D). Histidine 12 shows a n upjield shift of 10 Hz in the fully protonated form, and its pK is increased from 6.2 to 8.0. For histidine 119, there is a 20 Hz downfield shift a t low pH, and an increase in pK from 5.8 t o 7.4. Comparison of these curves with the pH-dependence of the binding constant of 3’CMP (Herries et al., 1962; Anderson et al., 1968) shows that the deprotonation of histidines 12 and 119 and the corresponding upfield shift of their C2-H resonances occurs only when the binding constant has fallen so that the enzyme is no longer saturated with 3’-CMP. I n the ribonuclease-3’-CMP complex, therefore, both these histidine residues must be protonated. Furthermore, the binding constant of 3’-CMP falls off riot only above pH 5.5 (on deprotoriation of the two histidine residues), but also below this pH; this decrease in binding with decreasing pH indicates that the dianionic form of 3’-CMP binds more strongly than the nionoanionic form (Meadows et al., 1969).

516

G . C. K. ROBERTS AND OLEG JARDETZKY

The downfield shift of the C2-H peak of histidine 119, 20 Hz beyond the normal chemical shift of the fully protonated residue, is similar to the shift of the C2-H peak of imidazole 011 formation of an imidazole-phosphate TABLEIX

Comparison of the Eflects of Cytidine .??I-, 3’-, and 5’-Monophosphates on Histidine Residues 12 and 115 of Ribonucleasea

His 12 Compound 2’-CMP 3’-CMP 5‘-CMP a

His 119

ApKb

ApKb

AAc

+ 10 +7 +10

1.8 1.8 1.8

A AC

- 25 - 20 0

72.2 1.6 1.2

Meadows et al. (1969).

* Change (increase) in ph’ in the presence of

the mononucleotide inhibitors. Change in chemical shift (in Ha a t 100 MHz; negative shifts are downfield) from the normal position for the fully protoriated residue.

I

I

5.0

I

I

I

6.0

PH

7.0

I

8.0

,

FIG.30. Titration ctirves of the histidine residues of ribonuclease A in the presence and absence (- - - - -) of 0.03 M 3’-ChIP. The ordinate is the chemical shift of the C2-H or C4-H peaks, in Hz (at 100 MHa) downfield from hexamethyldisiloxane. From Meadows and Jardetzky (1968). (-)

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

517

-

_____--_-_______-_-_--_~--3’-CMP CH , ____________________--------___--------3’- CMP C ’ H > _,_-----L - ----*-----.-_

640-

-

1

1

,_________-_____-__-___________,____,)----------~--

I

1

I

I

I

I

I

I

FIG.31. Chemical shifts [in Hz a t 100 MHz downfield from hexamethyldisiloxane (HMS)] of ribonuclease A aromatic peak and 3’-CMP pyrimidine C6-H and C5-H alld ribose C1I-H peaks as a function of total added 3’-CMP concentration. Ribonuclease concentration, 0.0065 M in 0.2 M NaCl (DzO); pH (meter reading), 5.5, 32°C. Dashed lines indicate positions of peaks of 3’-CMP alone a t equivalent concentrations. From Meadows and Jardetzky (1968).

complex in dimethyl sulfoxide (Cohen, 1968). This suggests that histidine 119 interacts directly with the phosphate group of the 3’-CMP. Support for this proposal comes from a comparison of the effects of 3’-CMP with those of the other cytidine mononucleotides, 2’-CMP and 5’-CMP (Table IX). It is apparent that histidine 119 is sensitive to the position of the phosphate group in the different mononucleotides, while histidine 12 is not. Thus it can be concluded that histidine 119 probably forms a direct bond to the phosphate group of 3’-CMP and 2’-CMP (but not to that of 5’-CMP, which causes no additional downfield shift of the C2-H peak of histidine 119). Histidine 12, on the other hand, has its pK markedly increased by the proximity of the negatively charged phosphate group but does not interact as specifically.

518

G . C. K . ROBERTS AND OLEG JARDETZKY COMPLEX OF 3’-CMP WITH RNASE

FIG. 32. Postulated structure of the 3‘-CMP ribonuclease complex, viewed from the back of the active site cleft. From Meadows et al. (1969). ENY LALANINE

Similar observations of the effects of 2’-CMP and 3’-CMP on the histidine residues of ribonuclease have been made by Ruterjans and Witzel (1969). Their interprctation, which is based on the idea of a hydrogen-bonded pair of imidazoles a t the active site (Witzel, 1963), differs from that of Meadows et al. (1969), who do not find the irregularities in the titration curves of histidines 12 and 119 reported by Ruterjans and Witzel (1969; see Roberts et al., 1969~). Figure 31 shows the changes in chemical shift of the pyrimidine ring C5-H and C6-H and the ribose C1’-H resonances of 3’-CMP on binding to ribonuclease; all these peaks shift downfield on binding (since the experiment was done a t a constant concentration of ribonuclease and increasing conceritration of inhibitor, the proportion of 3’-CMP bound increases froin right to left). I n addition, the upfield shift of a peak in the main aromatic envelope is shown; this peak has an area equivalent to about five protons, :tnd represents either a phenylalanine residuc or the upfield doublets of a t least two tyrosirie residues. The shifts of both the pyrimidine ring protons of the inhibitor and the aromatic group on the enzyme are identical for all three cytidine mononucleotides. I n the presence of phosphate or sulfate,

COMPLEX OF 5'-CMP WITH RNASE

CHz,

/CHz'CH,

CHZ LYSINE 41

'+ H/F\H H

FIG. 33. Postulated structure of the 5'-CMP-ribonidease complex viewed from the back of the active site cleft. From Meadows et al.

I19

(1969).

PHENYLALANINE 120

0-H

COMPLEX OF 2'-CMP WITH RNASE

119

FIG. 34. Postulated structure of the 2'-CMP-ribonuclease complex, viewed from the back of the active site cleft. From Meadows et al. (1969).

520

G . C. K. ROBERTS AND OLEG JARDETZKY

no change is seen in the aromatic region of the NMR spectrum of the enzyme, suggesting that the shift of the aromatic group is associated with the binding of the pyrimidine ring. The shift of the ribose Cl’-H resonance is approximately 3 Hz downfield in both 3’-CMP and 5’-CMP; however, when 2’-CMP binds to the enzyme, its C1‘-H peak is shifted 12 Hz downfield, indicating an appreciable difference in the orientation of the ribose moiety in this complex. From these NMR studies, the following conclusions were drawn: (1) The phosphate group of 3’-CMP and 2’-CMP binds specifically to histidine 119, while that of 5’-CMP does not. I n no case does the phosphate group bind preferentially to histidine 12. Both histidine 12 and 119 are protonated in the mononucleotide-enzyme complexes. (2) The pyrimidine ring of all three mononucleotide inhibitors binds in the same way, close to an aromatic group on the enzyme. (3) The ribose ring of the inhibitor is in a somewhat different orientation in the 2’-CMP-ribonuclease complex than in those with the other two nucleotides. By combining this information with the relative positions of the amion acids in the active site as determined by X-ray diffraction (Wyckoff et al., 1967; Wyckoff and Richards, personal communication), the proposed structures for the three enzyme-inhibitor complexes shown in Figs. 32-34 ;Yere obtained. I n the 2’-CMP complex (Fig. 34), if the phosphate group and pyrimidine ring of the nucleotide are to bind as the N M R evidence indicates, the orientation about the glycosidic bond must be s y n ; this explains the difference in the orientation of the ribose ring in this complex deduced above. The evidence for the structures shown is presented more fully by Meadows et al. (1969), and the mechanistic implications are discussed by Roberts et al. (1969a). The NMR spectra of ribonuclease TI in the presence and absence of inhibitors have also been reported (Inoue and Inoue, 1966). 6. L y s o z y m e . The binding of N-acetylglucosamine and its dimer and trimer to lysozyme have been studied by Cohen and Jardetzky (1968) and McDonald and Phillips (1968). I n each case, changes in the main aromatic envelope of the NMR spectrum were observed (Fig. 35) which appeared to be due to shifts of tryptophan resonances; it was concluded that the three oligosaccharides each occupied a part of the active site cleft close to one or more tryptophan residues. This is in agreement with the X-ray diffraction data on this enzyme (Blake et al., 1967). In addition, changes in the peaks a t the extreme high field end of the spectrum were seen; the peaks that change may represent mekhyl groups close to the tryptophans in the binding site. It is clear that, in the case of lysozyme, in which the resonances of the groups in the binding site could not be resolved, the amount of information obtained from the binding study was much less than in the case of ribonuclease.

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

(e)

k &V, 900

I 850 800 cps from HMS

521

I 750

FIG.35. Aromatic region of the NMR spectrum (100 MHz) of hen egg-white lysozyme (lo'% in 0.1 M NaCl/D20, pH 4.5) in the presence of increasing concentrations of triNAG (the 01-4 linked trimer of N-acetyl-D-glucosamine). (a) Lysozyme alone, timeaverage of 35 scans; (b) +0.5 molar ratio tri-NAG, 38 scans; (c) +1.0 molar ratio triNAG, 25 scans; (d) +5.0 molar ratio tri-NAG, 20 scans. The C2-H peak of the single histidine residue appears a t about 920 Hz (downfield from hexamethyldisiloxane). Thenumbers ident.ify regionsof the spectral envelope: 1, C3,5-H of Tyr; 2, C2,6-H of Tyr and C5,6-H of Trp; 3, C2-H of Trp and Phe; 4, C4,7-H of Trp; 6, C4-H of His. From Cohen and Jardeteky (1968).

c. Staphylococcal Nuclease. The problem of resolution can, as already indicated, be overcome by selective deuteration. This approach is well illustrated by the studies of thymidine-3',5'-diphosphate (pdTp) binding to staphylococcal nuclease (Markley el al., 1968a; Markley, 1969). The binding of pdTp was examined with analogs of staphylococcal nuclease deuterated so that the only resonances observed in the aromatic region were (1) those of the single tryptophan residue (2) single peaks (C3, 5H) for each of the seven tyrosine residues, and (3) the C2-H peaks of the four histidine residues. The effects of pdTp and Ca2+ (which is necessary for the activity of the enzyme) o n the chemical shifts of the tyrosine residues are shown in Fig. 36. Four of the seven tyrosine peaks are affected.

522

G. C. K. ROBERTS AND OLEG JARDETZKY

8.5

/-

1.21-

I

H3

-

I

I

I

1

I

I

I

I

i

>

6.9

c

I I

. ‘7

6.81 6’7

0

pd Tp - H 1’

I

0.’5

110 lj5 2.6 2.2 IpdTpl/lNase-D41 I

/+ I

I

I

I

I

FIG.36. Effects of adding thymidi~-3’,5’-diphosphate (pdTp) or Caa+and pdTp to a selectively deuterated analog of staphylococcal nuclease (Nase-D4). Assignments: His, H I 4 (imidazole C2-H) ; Tyr, Y 1-7; Trp, W (C2 ring proton) ; pdTp, H-6 (pyrimidine ring C6-H), and HI’ (ribose C1’-H). From Markley (1969).

When pdTp is added in the absence of Ca2+(Fig. 36), peaksY2and Y5shift upfield by 0.12 ppni and -0.2 ppm, respectively, and peak Y4 shifts downfield by -0.15 ppm. On addition of Ca2+to this pd‘rp-enzyme solution, at a molar ratio of one to one, peak Y5 shifts -0.35 ppm downfield. In the presence of both pdTp and Ca2+, therefore, peak Y5 is 0.15 ppm downfield of its positioii in the enzyme alone. Peak Y1 continues to shift upfield until a Ca2+:enzyme ratio of a t least 5.0 is reached. This indicates that the shift

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

523

of peak Y1 is produced by the binding of Ca2+to a second, weaker, binding site. None of the tyrosine peaks are affected by the addition of Ca2+alone. All the evidence is consistent with the proposal that peaks Y4 and Y5 are affected directly by the binding of the pdTp molecule. The upfield shift of peak Y5 in the absence of Ca2+could be due to the proximity of one of the negatively changed phosphate groups of pdTp, in which case the downfield shift on addition of Ca2+ may be ascribed to the neutralization of this negative change by a calcium ion binding t o the phosphate group. T h e change in chemical shift of peak Y4 is little affected by Ca2+, suggesting the possibility that the tyrosine corresponding to this peak is close to the ribose or pyrimidine moieties of the inhibitor. The involvement of three ph 5.5

O.2M NaC;

t

.

:

900

800

700

-1

cps from HMS

FIG.37. Aromatic region of the NMR spectrum (100 MHa) of ribonuclease A in the presence of increasing concentrations of Cu(I1) [0.1 to 1.2 X M ; ribonuclease concentration 0.006c5M , 0.2 M NaCl/DD, pH (meter reading) 5.5, 32"CI. The C2-H peaks [850-900 Hz downfield from hexamethyldisiloxane (HMS)] are, from left to right, those of residues 10.5, 12, 119, and 48. The top spectrum is essentially identical to the spectrum in the absence of Cu(I1).

524

G. C . K. ROBERTS AND OLEG JARDETZKY

tyrosine residues in the binding site is consistent with the suggestion made by Cuatrecasas et al. (1967a,b, 1968) on the basis of chemical modification and fluorescence spectroscopic studies. The model of the binding site of the enzyme derived from the NMR experiments is discussed in detail by Markley (1969). Although the number of detailed binding studies of this type which have been carried out is small, it is clear from these examples that, when the resonance lines of groups at the binding site can be resolved and, preferably, assigned, the quantity and quality of the information obtained by NMR is comparable only to that obtainable by X-ray diffraction. d . Binding of C u ( I I ) to Ribonuclease. The effects of paramagnetic species on protein spectra are a special case. The binding of Cu(I1) t o ribonuclease (Roberts and Jardetzky, unpublished) is an example of the use of selective broadening effects t o define the binding site(s) of a paramagnetic species. Figure 37 shows that, as the Cu(I1) concentration is increased, the C2-H peaks of histidines 105 and 12 broaden first, that of M. histidine 119 remaining unaffected a t Cu(I1) concentrations of 1.2 At higher concentrations, this peak, too, is broadened, and a t still higher concentrations (-lop3 M ) nonspecific broadening of the whole spectrum is observed. Thus Cu(I1) binds strongly to or near to histidines 105 and 12, and weakly to histidine 119. Contact and/or pseudo-contact shifts on addition of Co(I1) to lysozyme have been reported (McDonald and Phillips, 1969b), and selective broadening of histidine 12 and to a certain extent of histidine 119 was observed when a spin-labeled inhibitor was bound t o riboriuclease (Roberts et al., 1969b).

C . Protein Conformation and Conformational Changes 1. Overall Conformation: Denaturation

The dramatic changes produced in a protein NMR spectrum by denaturation of the protein have long been recognized (Saunders and Wishnia, 1958; Kowalsky, 1962; Mandel, 1964). More recent studies (McDonald and Phillips, 1967; Cohen and Jardetzky, 1968; Markley et al., 1968b; Bradbury et al., 1967a; Hollis et al., 1967) have shown that the NMR spectrum is a sensitive indicator of the overall conformation of the protein. The kvork of Cohen and Jardetzky (1968) showed clearly that the conformation of lysozyme in 8 A f urea a t 32" is distinct from the native conformation, from that in 0.1 M NaC1 at 75", and from the fully denatured conformation (observed in 8 M urea with excess mercaptoethanol a t 75"; see Fig. 10). The process of denaturation can thus be examined in considerable detail by NMR. All the spectral lines become narrower on denaturation, since the local environments and hence the chemical shifts of different residues

NMR SPECTROSCOPY OF AMINO ACIDS,

9.0

8.5

,s

8.0

PEPTIDES, AND PROTEINS

7.5

525

7.0

( PPm )

FIG.38. Aromatic region of the NhlIl spectrum of staphylococcal nuclease showing the changes on progressive t,hermal denaturation. All spectra pH 6.00, 0.3 M NaCl; (a) 32"; (b) 44"; (c) 47"; (d) 63". Assignments: His, H1, H2a, H2b, H3, H4 (imidazole C2-H) or H (low-field C2-H, high-field C4-H); Phe, F; Tyr, Y; Trp, W (C2 ring proton), and W' (C4,7 ring protons). The peak X-HMS is due to an impurity in the external standard. From Markley (1969).

526

G. C. K. ROBERTS AND OLEG JARDETZKY

of the same type become more nearly equivalent, and the motional freedom of segments of the polypeptide chain is increased; this is seen in Fig. 38, which shows the tnermal denaturation of staphylococcal nuclease as fol1968b; Markley, 1969). It is clear from lowed by NMR (Markley el d., these spectra that the four histidine residues become equivalent in chemical shift by 44", followed by the tyrosine and phenylalanine residues, while the peaks of the tryptophan residue do not reach their normal positions until 63". This suggests that denaturation in this case is not an all-or-none process, but proceeds by stages, partially denatured fornis having appreciable stability. McDonald and Phillips (1968) have observed double peaks for some resonances of lysozyme during thermal denaturation, suggesting a slow exchange between two states, presumably native and fully denatured. This has been interpreted as evidence for a single-step denaturation; further work is needed to settle the point. The denaturation of a selectively denaturated analog of staphylococcal nuclease a t alkaline pH is shown in Fig. 39 (Markley, 1969). In the native protein (pH < lO.O), the C2-I3 resonance of the single tryptophan residue (peak W) is about 0.51 ppm farther upfield than in the amino acid. When the enzyme unfolds, in the pH range 10.3-11.0, this resonance shifts abruptly downfield; a t pII 11.0, its position is identical to that in the free amino acid. In this pH range, the tyrosine C3,5-H peaks (Yl-Y7) will be expected to show shifts due to both denaturation and ionization of the phenolic .hydroxyl group. At pH 11.0, all the tyrosine resonances are equivalent, and the chemical shift is essentially identical to that of the free amino acid in the ionized form; the enzyme appears to be fully unfolded a t this pH. Cheniical shift changes in this pH range can be used to follow the process of unfolding, as well as to indicate the end points. I n the native protein, the resonance lines of some of the tyrosine residues are shifted appreciably from the position in the free, nonionized amino acid. Thus, while peaks Y1, Y2, and Y3 are within 0.05 ppm of the position of the free amino acid resonance, peaks Y4 arid Y5 are 0.14 ppm farther upfield, and peaks YG and Y7 are 0.31 ppm farther upfield. The last four peaks do not, therefore, show the full 0.26 ppm upfield shift due to ionization-indeed, the net shift of peaks Y6 and Y7 is 0.02 ppm downfield. The apparent pK of the tyrosine residues seems to be in the range 10.0-10.3. It is interesting to note that the chemical shift of the tryptophan C2-H resonance a t pH 10.3 is that found on the native protein, although the tyrosine residues are a t least half ionized. Either the overall unfolding of the molecule occurs a t pH 10.7, as indicated by the tryptophan chemical shift, in which case the fractional ionization of the tyrosine residues must he greater than 0.5 for unfolding to occur, or the unfolding takes place progressively, in the pH range 9.5-1 1.O, and the interaction responsible for the abnorinal chemical

527

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

7.8

I

1

1

I

I

I

I

7.7

7.5

2

-

W

“* 7.4

CL Q

In

L a

1 1

P

7.3 7.2

7. I 7.0

I 1

7.0

I

7.5

I

8.0

I

8.5

I

9.0

Ph *

I

9.5

I

10.0

I

10.5

I

11.0

FIG.39. Chemical shifts of the tyrosine (Yl-7) and tryptophan (W) aromatic protons of a selectively deuterated analog of staphylococcal nuclease in the pH range 7.011.0. From Markley (1969).

shift of the tryptophan C2-H resonance is among the last t o be disrupted in this progressive unfolding. A detailed investigation of the processes involved in the unfolding of proteins under various conditions is perhaps the most promising approach t o an understanding of the rules which govern the specific folding of these molecules. NMR is uniquely well equipped for such studies, since the protein can be examined in solution, and yet detailed information on the environment of individual residues can be obtained. Indeed, the “degree of denaturation” of every residue in the protein can, in principle, be defined and followed by NMR. Whenever some of the resonance lines can be assigned to individual residues-for example, the histidine residues of ribonuclease-these lines can be used to monitor the unfolding of specific sections of the polypeptide chain. I n ribonuclease, the histidine residues are a t positions 12, 48, 105, and 119 in the sequence of 124 amino acids, so that a good overall picture of the unfolding could be obtained in this way. As with other protein NMR experiments, the value of the information

528

G. C. K. ROBERTS AND OLEG JARDETZKY

obtained depends to a large extent upon the ability to resolve individual resonances and to assign them to particular amino acid residues in the protein. 2. Conformational Equilibria The combination of high information content with the dynamic nature of NMR measurements permits the study of conformational equilibria in proteins in unrivaled detail. The most clearcut example of such an equilibrium occurs in staphylococcaI nuclease (Markley, 1969; Markley et al., 1969). This enzyme, produced by the Foggi strain of Staphylococcus aureus, contains four histidine residues. However, the histidine C2-H region of its NMR spectrum contains five peaks a t most pH values. All these peaks change position with pH as would be expected of histidine C2-H peaks (Fig. 40), but two of them have appreciably less intensity than the others. The most likely explanation of these observations is that the histidine residue corresponding to peak 2 in Fig. 40 can exist in two states, representing two conformations of the enzyme, and that the exchange of the histidine residue between these states is slow on the chemical shift time scale (<24 sec-l). I n a fairly narrow pH range about pH 7, the two peaks broaden and coalesce to a single peak. Although the titration curves of the histidine residue in the two forms cross a t about this pH, the observed broadening of the two peaks must reflect a maximum in the rate of exchange a t about pH 7 ; the cause of this maximum is not yet clear. Since the histidine residue can be titrated independently in the two conformations, and the relative intensity of the two peaks is pH dependent, the equilibrium appears to be of the type E "+& *

E' H+&H

where Keq

=

[EI/[E']

>1

and K H= ~ [H+][E']/[E'H] < [H+][E]/[EH]= K H ~

Since addition of Ca2+ and/or thymidine-3',5'-phosphate changes the pK of this histidine in only one of the two forms (that with the lower pK, corresponding to E in the scheme above), it appears that these compounds bind to the erizymc in only one of the two conformatioris (see hIarkley, 1969; Markley et al., 1969). I n addition to this conformational equilibrium, a distinct conformational change occurs when the inhibitor thymidine-3',5'-diphosphate (pdTp) binds

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

529

FIG.40. Titration curves of the four histidine residues of staphylococcal nuclease. The major and minor peaks of H2 are indicated by 0 and 0 , respectively. From Markley (1969).

to this enzyme. This transition is manifested in the anomalous shape of the chemical shift curve of either Y2 or Y4 (Fig. 36). Since the two peaks overlap over part of the pdTp concentration range, there is some ambiguity in fitting the chemical shift data. It is quite clear, however, that it is not possible to fit simultaneously both Y2 and Y4 shifts to two simple mass action curves as it usually is in the absence of any interfering effects (see, e.g., Fig. 29). The most likely source of the discrepancy is a second conformational transition, involving a tyrosine residue (Y2 or Y4). This transition does not seem to occur in the absence of inhibitor, so that the overall scheme is E ‘ 6 E’ G E” (I) His

Tyr

530

G . C. K . ROBERTS AND OLEG JARDETZKY

A more complicated scheme, allowing for the existence of E’I and other species can also be written and may become necessary as more detailed data become available (Williams and Jardetzky, unpublished). A single conformational transition has been observed in pancreatic ribonuclease. I n the NMR spectrum of this enzyme, the histidine 48 C2-H peak is very broad (Meadows et al., 1967, 19GS). I n 0.2 iM sodium acetate, the peak can be followed throughout its titration range, but in 0.2 A1 sodium chloride, it broadens beyond detection above about pH 5 (Roberts et d., 1969~). Furthermore, the line width and the chemical shift of this peak are altered on inhibitor binding, even though histidine 48 is remote from the binding site. On this basis it has been suggested (Meadows and Jardetzky, 1968; Meadows et al., 1969) that there exists a conformational equilibrium affecting histidine 48 and that the position of this equilibrium is altered by inhibitor binding. It is possible that the conformational transition involved is the same as that detected by French and Hammes (1965) by the temperature-jump method. French and Hammes (1965) proposed that the equilibrium was of the form k+l

EH’ e EH e E

+H

and estimated k+l as 183 sec-’ in D2O a t 25’. From the intermediate exchange condition, the NMR data give an order-of-magnitude estimate of 60 sec-’ (D20,32’). The nature of the conformational change is discussed more fully by Meadows et al. (1969). 3. “Heme-Heme Interactions” and Other Studies of Heme Proteins

It is well established that hemoglobin undergoes a conformational change on oxygenation (Haurowitz, 1938; Perutz et aE., 1968). I t is also apparent that this change proceeds in stages; the binding of oxygen to hemoglobin is cooperative, i.e., the affinity of hemoglobin for oxygen increases with each successive molecule of oxygen bound. The structural basis of this cooperativity (the “heme-heme interaction” of earlier literature) has recently been the subject of a detailed study by R. G. Shulmari arid his colleagues (Shulman et al., 1969a,b,c; Wuthrich et al., 1968a,b, 1969; see also Kurland et al., 1968), using both high resolution NMR and electron paramagnetic resonance. The essential question raised in this irivestigation was whether the binding of an oxygen molecule to the heme group of one of the four chains of hemoglobin would result in a change of the electronic structure of the heme on a neighboring chain. Such a change would manifest itself as a change in the free energy of binding of a second oxygen molecule and could account for part or all of the observed differences in affinity. This difference could also be accounted for, in part or in its entirety, by a conforma-

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES,AND PROTEINS

531

tional change of the protein chain, not involving the heme group. The overall observed free energy difference reflecting the cooperative effect may thus be regarded as consisting of two parts (Shulman et al., 1969a): AFr = AFIH

+ AFIP

where AFr (approx. 3 kcal/mole) is the total observed free energy of interaction and AFIH and A F I P are the free energy contributions of the heme group and the protein chains, respectively. I n principle, high resolution NMR can provide sufficiently detailed information to evaluate both terms; however, the studies carried out thus far bear directly on the magnitude of the first term only. The reason for this is that the porphyrin spectrum lies outside the main protein envelope. Individual lines are readily observed and in some cases can be assigned. On the other hand, resonance lines of individual amino acid side chains cannot for the most part be resolved. I n an extensive preliminary study, Wuthrich et al. have made several assignments of the porphyrin resonances in sperm whale cyanometmyoglobiri (1968a), human cyanomethemoglobin (1968b), and a series of porphyrin iron (111) cyanides (1969). The assignments were based on the integrated intensity of the spectral lines, a comparison of the spectra of porphyrin with different side chains, and differences in the temperature dependence of chemical shifts. Observation of a marked temperature dependence for most lines led to the conclusion that the shifts originated in a contact coupling with the iron atom (cf. Section II1,C). Ring current shifts were ruled out as an alternative, since such shifts are temperature independent for protons attached to the ring. However, ring current shifts are solely responsible for the porphyrin lines in oxyhemoglobin and oxymyoglobin, since these compounds are diamagnetic and do not show contact shifts. The temperature dependence of contact shifts is given by (Bloembergen, 1957b) : Av,

=

-A

2

i;Hl

S(S+ 1 ) L 3k T

where Av, is the contact shift, A is the hyperfine coupling constant, ye and Y H the gyromagnetic ratios of the electron and the proton, respectively, S the electron spin, v the spectrometer frequency (220 mHz in this case), k the Boltzniann constant, and 7’ the absolute temperature. It is therefore possible t o calculate the hyperfine coupling constants from a plot of Av, vs 1/T. The observed values can be correlated with those expected from semitheoretical arguments for protons in different positions on the ring, leading to a self-consistent set of assignments. A set of A values has been

532

G . C.

K.

ROBERTS AND OLEG JARDETZKY

obtained for several porphyrins, cyanometmyoglobin and cyanomethemoglobin, and correlated with ?r-electron densities in the ring, using the McConnell relation (McConnell, 1956):

A

= QPC"

where Q = -6.3 X lo7 is a constant of proportionality (7.5 X 107 was considered a better approximation for porphyrins) and pc* is the ?r-electron spin density expressed as percent of one unpaired electron. Preliminary Huckel molecular orbital calculations seem to indicate that it will be possible to account theoretically for all the observed features of the porphyrin NMR spectra. This in turn should lead to an understanding of the interesting differences in affinity exhibited by different porphyrins for oxygen and other ligands. Experiments bearing more directly on the question of cooperativity were of three different kinds. First, a comparison of the spectra of the isolated CY and 0 chains to those in the tetramer showed that the porphyrin resonances were different in the three cases, indicating their sensitivity to conformational changes. Second, a careful comparison of the main spectral envelopes of deoxyhemoglobin and oxyhemoglobiri revealed some ill-defined but definite differences. In particular, a peak on the high-field side of the aliphatic envelope could be resolved in the oxygenated, but not in the deoxygenated forni. This was taken to indicate that a conformational change affecting the polypeptide chain did occur, even though its nature could not be specified. Finally, spectra of the porphyrin region (H20)P1'(02)]2, were compared for mixed state hemoglobins, particularly [a111 which was formed by combining oxidized CY chains derived from human oxyhemoglobin A and 0 chains derived from human oxyhemoglobin H, and a similarly formed [arl(Oz)P1rl(H~O)]~. The direct experiment, comparing [aI1(O~)fi1I(H20)]2 to [a11(H,0)011(H20)]2, is of course impossible because of rapid exchange. The important finding was that the observed mixed state spectrum was in all cases a simple sun1 of the spectra of the constituent chains. A similar finding resulted from parallel electron spin resonance experiments. The conclusion could therefore be drawn that the electronic structure of a heme group was not affected by the oxygenation of a neighboring heme; that AFIII 0, AFI = A F I p and "heme-heme" interaction, taking the term literally, did not exist. This same conclusion had been reached by Pauling (1949) and by Coryell et al. (1939) on the basis of spectroscopic and magnetic moment measurements. The origin of the cooperative effect must be sought in the rearrangement of the polypeptide chains themselves, probably at or near the interfaces. With the determination of the deosyheinoglobin structure a t 2.8 A resolution (Perutz, 1969), the extent of the structural rearrangenlent can be precisely defined. Its

=

N M R SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

533

dynamics remain to be described. Preliminary observations of the histidine region of the hemoglobin NMR spectrum (M. N. Williams, N. Greenfield, and 0. Jardetzky, unpublished data) indicate that it should be possible to do so, a t least in part.

D . Probe Experiments The principle of a probe experiment, whether using NMR, ESR, light absorption, or fluorescence, is to measure one or a t most a few easily accessible parameters and draw inferences about more complex phenomena that are more difficult to observe directly. Any such probe experiment represents an essentially monoparametric use of the method and is, in the case of NMR, inherently incapable of providing as much information as a complete analysis of a protein NMR spectrum. Nevertheless, probe experiments can give valuable information under conditions that do not allow the observation or analysis of the entire spectrum. Undoubtedly the most elegant and useful NMR probe technique is that pioneered by Mildred Cohn and co-workers, in which the relaxation rates of the solvent water protons are monitored in the presence of a paramagnetic ion, a protein to which it binds, and/or substrates or inhibitors. Depending on the exact conditions, such an experiment can give information on changes in the number of water molecules in the first coordination sphere of the protein bound metal ion, their relative motional freedom, and/or their exchange rates (see Cohn, 1967). This approach has provided a substantial amount of information about enzymes, such as kinases, which require paramagnetic metal ions. However, it will not be reviewed here since a detailed review will be published elsewhere (Mildvan and Cohn, 1969). I n another kind of NMR probe experiment, use is made of the fact that the relaxation rates of nuclei with quadrupole moments (spins greater than 1/2) are strongly dependent on changes in the electric field gradient at the nucleus and are therefore very sensitive indicators of binding. This application of NMR was originally suggested by Wertz (Wertz and Jardetzky, 1956; Wertz, 1957) and explored in the study of 23Nacomplexes (Jardetzky and Wertz, 1956a,b, 1960). Most of the recent experiments of this type have employed W 1 (e.g., Stengle and Baldeschwieler, 1966). The sulfhydryl groups of proteins (notably hemoglobin) (Stengle and Baldeschwieler, 1966, 1967; Ellis et al., 1969) and the helix-coil transition of a sulfhydryl-containing polypeptide (Bryant, 1967) have been investigated by W 1 NMR in the presence of mercuric chloride. 36ClNMR has also been used to study the binding of a mercury-containing hapten to an antibody (Haugland et al., 1967), and the binding of a mercury-containing inhibitor to chymotrypsin (Marshall, 1968). The mercury atom, bound to both the macromolecule and the

534

G . C. K. ROBERTS AND OLEG JARDETZKY

I

I!

H 60cps

i

lB

FIG.41. 35C1chloride ion riiiclear magnetic resonance, (4.33 MHz) for (A) 2.0 M NaC1-0.05 M phosphate, pH 7.0; (B) as A, plus 0.2% a-chymotrypsin; (C) as B, plus 1.0 equivalent, p-mercuribenzeriesulforiyl fluoride; (D) as C, biit enzyme pretreated with 20 equivalents of benzyl bromide. From Marshall (1068).

chloride ion, amplifies the effects of the macromolecule on the relaxation rate of the chloride ion because of the very large distortion of the electric field gradient which it produces a t the chlorine nucleus. It thus increases the sensitivity of the method by several orders of magnitude. For example, the line width of the 35Clresonance of 2.0 M sodium chloride is approximately 17.5 Hz (Fig. 41A; Marshall, 1968) arid is unaffected by the addition of 0.2% chymotrypsin (Fig. 41R). However, when one equivalent of the inhibitor p-mercuribenzenesulfonyl fluoride is added to the enzyme, the 35C1line-width increases to about 32 Hz (Fig. 41C); since the 35Cl ions are exchanging between the bound and free states a t a rate which is rapid on the NMR time scalc (see Table VII above), the observed linewidth is the weighted average of the line widths in the two environments. When the enzymc is pretreated with beiizyl bromide, which modifies a single inethionine residue a t the active site, the 35Clline width in the presence of one equivalent of p-mercuribenzene sulfonyl fluoride is still greater (-41 Hz) indicating more restricted motional freedom of the bound chlo-

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

535

ride ion (Fig. 41D; Marshall, 1968). Binding of chloride ion to horse radish peroxidase (in the absence of mercury) has been observed, using 35C1 KMR, by Ellis et al. (1969). Ward (1969) has observed the interaction of chloride ions with the zinc atom in bovine carbonic anhydrase by the same technique. The zinc-chloride interaction is highly pH dependent, decreasing to zero above pH 10. The apparent pK, of the broadening in W1 line width, produced by the enzyme, depends on C1- concentration; it is approximately 7.0 when extrapolated t o (Cl-) = 0. The zinc-chloride interaction is blocked by cyanide and acetazolamide. The deuterium relaxation method has been used to study protein hydration (Glasel, 1968); its usefulness, in conjunction with specific deuterium labeling, for polynucleotide studies (Glasel et al., 1968) suggests that it may prove to be valuable also in the study of proteins. A precise molecular interpretation of probe experiments is of course contingent upon detailed knowledge of the structure of the binding site.

VI. CONCLUDING REMARKS The foregoing discussion has been devoted to but a few of the potential applications of NMR to protein chemistry. However, even the limited number of studies that have been reported have established beyond any doubt the power of the method and the wide range of problems t o which it is applicable. At the time of this writing, the field has barely emerged from the stage a t which the reproducibility of the data and methodological improvements were the major concern. Without sensitivity enhancement by time averaging (Klein and Barton, 1963; Jardetzky et al., 1963), meaningful protein NMR spectroscopy was beyond reach. With the advent of more sensitive spectrometers (see McDonald and Phillips, 1967),and in particular of Fourier transform NMR spectroscopy (Klein, 1968a,b; Vold et al., 1968; Ernst, 1966), the collection of data is no longer a factor limiting experimental design. With the success of selective deuteration of proteins (Markley et al., 1968a,b), the two major problems of proteinNMRspectroscopy-resolution and assignment of resonance lines-have been shown t o be soluble a t least in principle. It is t o be expected that future difficulties in overcoming these problems will be proportional to the complexity of the protein. Success, however, is t o be sought in experimental ingenuity rather tthan through further advances in instrumentation. A comparison between X-ray diffraction and high resolution NMR is of some interest because they are the only two methods that have a sufficient information content t o define the structure of a protein. Both methods are nondestructive. The information obtainable by NMR in the foreseeable future does not approach that derived from X-ray diffraction in geometric

536

G. C. K. ROBERTS AND OLEG JARDETZKY

precision: NMR does, nevertheless, have several unique features. It allows the observation of a protein in solution. It makes it possible to follow the time course of conformational transitions, t o distinguish between different conformational changes occurring simultaneously, and to define conformational equilibria. It permits the deterniination of the charge state of an individual amino acid residue and the identification of a kinetic measurement with a specific structural change. It allows direct detection of an interaction between neighboring groups, whereas X-ray diffraction allows only an inference based on proximity. I n the solution of such problems as enzyme catalysis, folding of polypeptide chairis, and mechanisms of drug action, the information derived from the two methods may thus be regarded as complementary. The uniqueness of the information obtainable by high-resolution NMR makes it, along with X-ray diffraction, one of the two most powerful structural methods available to modern protein chemistry.

APPENDIX:BASICCONCEPTS

A brief qualitative description of the nuclear magnetic resonance experiment can be given most easily in terms of a single nucleus, although in practice it is necessary to consider an assembly of nuclei. If a single magnetic dipole is placed in a magnetic field, Ho, a torque will be exerted on it which will tend to orient it in the direction of the applied field. In the absence of any other forces, the dipole will become completely aligned with the field Ha. However, if the magnetic dipole is that of an atomic nucleus, it is associated with a spin angular momentum by virtue of the rotation of the nucleus about its own axis. The addition of these two motions (i.e., the spin and the alignment of the dipole with the field Ha) leads t o a resultant steady rotation of the dipole (and its spin axis) about the direction of the applied field Ho. For given type of nucleus, e.g., a proton, the frequency of this precession depends only on the magnitude of the local magnetic field a t the nucleus. The angle formed by the magnetic moment vector of a nucleus with the direction of the field is uniquely determined by the quantum rules, so that each nucleus can precess about the field Ho in only a few discrete orientations. For nuclei, such as the proton, with a spin quantum number of $5, there are only two possible orientations. Each orientation corresponds to 4 In principle, reasonably precise iriformatiori on interatomic distances could he obtained from relaxation measurements on stcreospecifirally deuterated polypeptide chains, and conformation of individual residiies could be defined from measurements of coupling constants. The technical difficiilties of siich an undertaking are, however, a t least for the present, prohibitive.

NMR SPECTROSCOPY OF AMINO ACIDS, PEPTIDES, AND PROTEINS

537

a particular energy level of the nucleus; for nuclei of spin 46, there is thus a ground state and a single, higher energy, excited state. If we now introduce a second field, HI, much weaker than Ho,and rotating in the plane perpendicular to Ho, this will exert an additional, continuously varying, torque on the nucleus. Since H1 is much weaker than Ho, this torque will be negligible, except when the frequency of rotation of H1 is exactly equal to the frequency of precession of the nuclear dipole about the field H,. Under this condition, the torque produced by H1 will have a constant value and will cause a reorientation of the dipole with respect to the field Ho. The energy required for this reorientation, or transition, to the excited state is equal to the difference in potential energy between the ground and excited states, and is absorbed from the rotating field HI. The frequency of rotation of H I a t which energy absorption (resonance) is observed is therefore equal to the precession frequency of each nucleus, which depends upon the local field a t the nucleus. The field H1 is provided by the rotating magnetic vector of plane or linearly polarized radiofrequency electromagnetic radiation, generated so that its magnetic vector oscillates along an axis perpendicular to H,. It is the absorption of energy from this radiation that is actually observed as the resonance signal. The characteristics of this signal are defined as follows: 1. Chemical shift. The position (resonance frequency) of a resonance line. The presence of an external magnetic field induces electronic currents in matter, giving rise to induced diamagnetic moments which tend to oppose the applied field. As a result, in diamagnetic substances the effective field, Heffla t a given nucleus will be somewhat lower than the applied field Ho, and the nucleus is said to be shielded. The magnitude of this shielding can be expressed by an equation of the form, where u is a dimensionless parameter known as the shielding constant, which is a sensitive function of the local electronic environment. The position (resonance frequency) of a line is thus a measure of the shielding constant for the particular nucleus. Any nuclei of a particular type (e.g., 'H) in a sample which are in identical environments will experience identical local magnetic fields, and will thus have identical resonance frequencies. Such nuclei are said to be magnetically equivalent and, taken together, constitute an assembly known as a magnetically equivalent set. Because of the problems involved in obtaining accurate measurements of field strength, an absolute scale is impractical, and line positions are measured relative to a standard, either internal (in the same solution as the

538

G . C. K. ROBERTS AND OLEG JARDETZKY

sample) or external (oftenin a coaxial capillary tube). The most commonly used standards are tetramethylsilane (TMS), hexamethyldisiloxane (HMS), arid 2,2-dimethylsilapentane-5-sulfonicacid (DSS). Thus, in frequency units A v = vs - v,

(in cycles per second)

(A2)

where A v is the chemical shift, v, is the resonance frequency of the sample and v, that of the reference. To facilitate comparison of measurements made a t different field strengths (and hence a t different oscillator frequencies), the chemical shift is also often expressed as a dimensionless number 6, where 6

Av

= -X Yasc

106 (in parts per million)

(A31

Since most protons have resonance frequencies below those of TMS, these measures of chemical shift are strictly negative numbers. However, they are frequently quoted as positive numbers with the designation “downfield from TMS.” The alternative widely used in organic chemistry is the T scale, defined by T = (10.0 6) ppm. 2. Coupling constants. In any atom, the magnetic field of the nucleus will tend to orient the spin of its valence electron antiparallel to the nuclear spin. At the same time, the spins of the two valence electrons in a covalent bond must be paired with each other, so that the valence electron on the neighboring atom will tend to have its spin oriented parallel to the spin of the nucleus (N,) in the first atom. As a result a magnetic field is created at the second nucleus (N2). Thus (for nuclei of spin 1/2) each spin state of nucleus NP is split into two states, each corresponding to one of the two possible spin states of nucleus NI. Since a n identical effect is transmitted through the same bonding electrons from nucleus Nz to nuclcus N,, the absorption of NI will be similarly be split. In general, if a nucleus is coupled in this fashion to n nuclei of spin I, its absorption line will be split 1) components. The coupling constant, J N I N 2 ,is a measure of into ( 2 n I the strength of the coupling between two nuclei; it depends on the electronic configuration (hybridization) of the bond between them, and on the relative orientations of the two nuclear spin vectors, but not on the strength of the external magnetic field. Coupling effects generally extend over no more than four or five covalent bonds. If the chemical shift difference between two nuclei, AvN,N>, is much greater than the coupling constant between them, the value of thc coupling constant can be obtained directly from the spectrum; if A V N , N ~5 J N I N P ,a more complex analysis is required, but the values can still be obtained.

+

+

NMR SPECTROSCOPY O F AMINO ACIDS, PEPTIDES, AND PROTEINS

539

3. Relaxation time. Just as an external magnetic field oscillating a t the resonance frequency can produce transitions of nuclei between two spin states, so can oscillating local fields. These transitions induced by local fields provide a mechanism for a loss of potential energy from any given nucleus. This is referred to as relaxation. If the local field producing relaxation arises from other nuclei in the same magnetically equivalent set, then two nuclei within the set will exchange states and there will be no loss of potential energy from the set as a whole, but there will be a decrease in the net magnetic moment of the set because the nuclei lose phase coherence as a result of the exchange. This is known as spin-spin (or transverse) relaxation. Exchange of energy with an oscillating local field arising from any other source will result in a net loss of potential energy from the set; this is known as spin-lattice (or longitudinal) relaxation. The local fields producing spin-lattice relaxation can arise from many sources, including other nuclei on the same molecule, nuclei on other molecules and electrons. The two relaxation processes are described by two characteristic relaxation times, T I (for spin-lattice relaxation) and Tz (for spin-spin relaxation). The relaxation time Tz can be interpreted as the average time required for two nuclei to lose phase coherence in their precession about the external magnetic field as a result of spin-spin relaxation. It is related to the width of an NMR absorption line by the equation

where Avlla is the full width of the line a t half-maximum height. Thus the shorter T z (the more efficient the relaxation process), the broader the line. In the absence of local field inhomogeneities (which, in liquids and gases, are averaged to zero over time), spin-spin relaxation tends to be slow. In liquids, the dominant relaxation mechanism is spin-lattice relaxation, which proceeds through the dipolar interaction of the nucleus with that component of the local field (originating outside the equivalent set) which fluctuates a t the resonance frequency. As energy is given off to this local field, a transition from a higher to a lower spin energy level occurs with a time constant TI. Each of these transitions necessarily leads to a change of phase, so that in liquids in the absence of chemical exchange T1 = Tz. The oscillating component of the local field will in turn decay with a time constant T ~ the , correlation time. This can be interpreted as the time during which two nuclear or electronic dipoles maintain a given orientation in space with respect to one another. Thus the slower the rate of atomic reorienta, shorter 2'1 and the broader the line. I n liquids of tion, the longer T ~ the low viscosity, the correlation time, T ~ generally , depends on the rate of molecular tumbling (Brownian motion).

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