Carbon-13-NMR of peptides and proteins

Progress

in N MR

Spurrrmcopr.

1978.

Vol.

I?.

pp.

140.

Perpamon Press. Prmted m Great Britain

CARBON-13-NMR

OF PEPTIDES

AND PROTEINS

OLIVER W. HOWARTH Department

of Molecular Sciences, University of Warwick, Coventry CV4 7AL, England and

DAVID M. J. LILLEY G. D. Searle and Co. Ltd., Research Division, P.O. Box 53, Lane End Road, High Wycombe, Bucks, HP12 4HL, England (Manuscript received 28 March 1977)

CONTENTS 1. Introduction 2. Sensitivity Improvement 2.1. Sample Size 2.2. Magnetic Field Intensity 2.3. Noise-folding 2.4. Pre-amplifier Modification 2.5 Proton-enhanced ‘“C NMR 2.6. An Experimental Example 3. Chemical Shifts 3.1. Factors that Affect Chemical Shifts 3.1.1. pH 3.1.2. Through-space shielding 3.1.3. Solvent 3.1.4. Cis-tram isomerism in proline 3.15. Other identifiable conformational changes 3.1.6. Ion binding 3.1.7. Paramagnetic shifts 4. Coupling Constants 5. Spectrum Assignment in Peptides 6. Relaxation Times in Peptides 7. Proteins 7.1. Relaxation Times, I1 and T2, in Proteins 7.1.1. Factors affecting us 7.1.2. Factors affecting ho 7.2. Structural Shifts and Assignment of Resonances 7.2.1. Fitting to peptide shifts 7.2.2. Selective enrichment 7.2.3. Natural abundance single carbon resonances, including paramagnetic effects 7.3. Progress Towards Structure Determination in Proteins 7.3.1. Elastin ’ 7.3.2. The histones 7.3.3. i3C enrichment 1.3.4. 13C adducts 7.3.5. Semi-synthetic i3C incorporation 7.3.6. General enrichment 7.3.7. Specific enrichment 7.4. Ligand-binding Studies 7.4.1. Enzymes 7.42. Lectins and carrier molecules 7.4.3. Antibody-hapten interactions 7.4.4. A warning References 1.

INTRODUCTION

Carbon-13 nuclear magnetic resonance offers in principle many advantages over proton magnetic resonance for the study of peptides and proteins. The spectra are considerably easier to assign, with good chemical shift separation. they are less sensitive to

1F.\.\I.R.S I2 I---\

1 2 2

8 8 8 8 9 9 10 10 11 14 15 17 18 22 22 23 24 29 29 29 31 31 32 32 32 33 33 35 35 35 35

the presence of impurities, their linewidths and relaxation times give a direct measure of localised intramolecular motions, and they can readily be used in biosynthetic investigations because of the possibility of substantial selective isotopic enrichment. However, although ‘H nuclear magnetic resonance has received wide attention as a method for investigating proteins

2

OLIVER W. HOWARTHand DAVID M. J. LILLEY

and peptidesc1-5’ the use of 13C-NMR was relatively scantily reviewed(24) until very recently@’ (whilst this review was in preparation). The present review attempts to cover the literature up to the end of 1976; it does not repeat all the details of the accounts of specific molecules, including comparisons with ‘H-NMR results, given in reference (6), but attempts to present an overall view of the progress in, and possibilities offered by, the ‘3C-NMR of peptides and proteins, including relatively recent studies which increasingly tend to produce results of real biochemical and biophysical significance. The reason for the delayed interest in 13C-NMR of peptides and proteins is undoubtedly its relatively low sensitivity in comparison to ‘H-NMR, so that much early 13C work has involved the use of physiologically meaningless concentrations or quantities of sample that are not normally obtainable, However, recent instrumental advances are beginning to relieve these constraints, and even to open up the possibility of whole-tissue studies.‘7,8) Meanwhile, much of the earlier work has now laid an experimental and theoretical foundation for future studies. As instrument development is a major key to future advance, it will be treated first. 2 SENS~VI~

IMPROVEMENT

abundance 13C nuclear magnetic Natural resonance requires, by virtue of its lower magnetic moment and 1.1% natural abundance, an instrumental sensitivity of 5700 times that required for the equivalent proton spectrum. This order of sensitivity was not generally attainable for solutions until the advent of Fourier transform NMR, which permits rapid signal accumulation. Another necessary innovation was efficient wide-band heteronuclear ‘H decoupling, which uniformly reduces spectrum complexity and increases the sisal-to-noise ratio both by narrowing and coalescence of multiplet resonances and also via the nuclear Overhauser enhancement (NOE). This latter is discussed quantitatively in a later section. With these advances alone, it was possible to study amino acids and many small polypeptides without isotopic enrichment, and just possible to obtain useful spectra from saturated solutions of favourable proteins such as lysozyme(9’ using spectrum accumulation times of about one day. Instrumental improvements such as automated block-averaging”” or a phi-alternating pulse sequence have made accumulations of this length routine by removing limitations imposed by the computer word size. However, 1-2 days still represents an approximate upper limit on accumulation time because of limitations of instrumental. stability and availability. Thus recent advances in sensitivity have had to come from other developments, which are now discussed individually. 2.1. Sample Size 13C NMR, in particular that of proteins, has a relatively modest magnetic field homogeneity require-

ment. Even in mobile solutions, linewidths are often over 1 Hz, and in native proteins linewidths of 20 Hz or more are common for the resonances of carbons directly bound to protons. The earliest 13C-NMR spectrometers took advantage of this to the extent of using 8mm or 10mm od. sample tubes, with a typical minimum sample volume of 0.8 ml. By careful design using conventional magnets it has proved possible to reach sample tube diameters of 15 or 18 mm (2.1 T magnet) or 20 mm (1.4 T)“” this being the maximum that can be accommodated between the pole-pieces. Superconducting magnets permit even larger tube diameters; for example, the new Bruker WH180 WB spectrometer uses 25 mm o.d. tubes, i.e. a min~um 14 ml of solution. The observed signal-tonoise ratio, S/N, gain in these spectrometers is at least as large as the sample volume. Even if this volume of sample is achieved only by dilution of a saturated solution, there may nevertheless be a signal-to-noise gain because of increased molecular mobility and hence reduced linewidth. Furthe~ore, the more dilute solution may be closer to realistic physiological concentrations. 2.2. Magnetic Field Intensity In theory, the inherent sensitivity of a NMR experiment should increase as the square of the static induction field B,,, In practice, however, the observed gains have been considerably less than this for a number of reasons. The radiofrequency coil shape at higher frequencies is often less ideal, as very few turns are needed. The coil configuration in su~r~nducting magnets presents special difficulties. With narrow resonances, the disadvantage of a lower field may be offset by higher homogeneity. With resonances that are broad because the tumbling correlation time, T, is comparable to the Larmor frequency, o, the NOE will be less at higher w. Also the spin-lattice relaxation time, Tl, may well become inconveniently long at high fields (see Fig. 5). Thus, further increases in B. will only bring gains if there are also parallel advances in coil design. (12) Hoult and Richards have recently given a useful discussion of the problems of coil design.” 31 With very high fields and very slowly tumbling molecules, a further deleterious line broadening may arise because of chemical shift anisotropy. This broadening increases as B& and has already proved serious in the 31P-NMR of phospholipids at 129 MHz.““*‘~’ However, although detectable, it is not yet a serious problem in 13C-NMR. Against these disadvantages one must set a major advantage of using a large Bo. The chemical shift separation increases in proportion to Do, thus improving the resolution. In addition, the tumbling rate of many proteins is such that the spin-spin relaxation time, Tz. for many resonances will be appreciably longer at higher fields--typically it may double for a threefold increase in field. Thus high fields offer a much improved chance of seeing single carbon resonances,

Carbon-13-NMR of Peptides and Proteins

60

,

60

20

0

FIG. 1. (Upper) Aliphatic region of ribonuclease IIA “C spectrum at 67.9 MHz. (Measurement by courtesy of W. Hull, Bruker Spectrospin A.G.) (Lower) The same at 23.6 MHz.

even in the aliphatic regions of protein spectra. An example, native ribonuclease-A recorded at 67.9 MHz, is shown in Fig. la; although the signal-to-noise ratio is not remarkable (the solution was only 2m~), the resolution is almost an order of magnitude better than the corresponding 23.6 MHz spectrum in Fig. lb. The highest 13C frequency so far reported in use is 90.5 MHz.“@

eliminate almost all these higher frequencies. The removal of fhe negative frequency w. - w is harder, requiring either rather expensive quadrature detection or some skill in electronic modification”‘) such as the insertion of a pretuned quartz RF filter, set for fixed ~r)~,at an appropriate point in the detector circuitry. Commercial versions of such filters are just coming on to the market.

2.3. Noise-folding One disadvantage of digital Fourier transform technology is that the analogue-to-digital converter is unable in principle to distinguish contributions from the true detected audiofrequency o - w0 (where w0 is the chosen detector frequency) from the “folded” frequencies 2n(SW) f (w - w,,) where n is any integer and SW the chosen spectral width.“” Whilst it is easy to arrange that SW covers the resonances of interest, it is much harder to prevent the folding of noise, which thus increases as the square root of the number of frequencies, that are superimposed. Frequencies of magnitude greater than SW can be largely removed by appropriate audio filters; if it is convenient to make SW some 50% greater than the minimum width necessary to span the spectrum, then by careful adjustment of w. and of the filters one can actually

2.4. Pre-ampl$er Modijication Maurer and colleagues(‘s) have shown recently that a S/N gain of about three could be obtained on their 270 MHz spectrometer by a careful additive modification of the first RF simplification stage, involving the use of special low-noise transistors. However, it is not yet clear whether this substantial improvement can be extended readily to other instruments. Further improvement in detector design probably offers the best long-term hope for increased sensitivity. An ultimate goal, as yet technically out of reach, might be to supercool the entire receiver coil and first-stage amplifier, thus reducing Johnson noise almost to zero. This technique is already used by radio-astronomers, but would present major thermal insulation problems in NMR.

OLIVERW. HOWARTHand DAVID M. J. LILLEY

4

HEN EGG-WHITE LYSOZYME

TRP-62.63 C’

P”E

t TRP

I

I TRP C’

Regions of aromatic carbons and c’i of arginine residues in the convolution-difference naturaal abundance 13C Fourier transform NMR spectra of hen egg-white lysozyme. Each spectrum was recorded at 15.18 MHz under conditions of noise-modulated off-resonance proton decoupling, using 8192 time domain addresses, a spectral width of 3787.9 HZ, 49,152 accumulations, and a recycle time of 2.205 s (30 hours totaI time). The convolution-difference procedure was used with T, = 0.72 s, r2 = 0.036 s and K = 1.0. Peak numbers are those of Table 1. The insets (Peaks 1 to 6) are shown with one-eighth the vertical gain of the main spectrum. Assignments are given in the text. A, 14.6 mM protein in H20, pH 3.05, 0.1 M NaCI, 44”. B, 13.8 mM protein in D20, pH meter reading 3.08, 0.1 M NaCI, 42”. FIG. 2. 14.1 MHz *jC spectrum of lysozyme (aromatic region) processed to show only unprotonated carbon resonances (from reference 21). 2.5. Prato~-enhanced 13C NMR The resonances of solids or of very immobile liquids are generally broad and hence difficult to observe. However, Pines, Gibby and Waugh”‘) have developed a pulsed NMR technique called “proton-

enhanced NMR” in which successive proton spin population changes are used as a “pump” to create a greatly enhanced 13C population difference, and hence a much improve sensitivity. The technique remains a specialised one at the moment, but its potential has been demonstrated recently by the observation of the 13C resonance of labelled cholesterol dissolved in relatively immobile phospholipids.“” 2.6. An Experimental Example Figure 2”” shows an excellent pit-s~ctrum from a 20 mm o.d. sample of 14.6 mM lysozyme in 0.1 M NaCl, pH 3.1, 44°C. The spectrum is the average of 49,152 accumulations made at 14.6 MHz over about 5 h. Filtering was used to prevent all types of noisefolding, and a simple convolution-difference technique

was used to remove broad lines. The rem~ning resonances arise mostly from single aromatic and arginyi carbon atoms in the molecule. If all possible additional existing technology could be applied to this measurement, a further sensitivity gain of around four might be achievable, provided some 20 ml of sample could be found, i.e. about 4 g of lysozyme. However, many useful results could be obtained at much lower concentrations. 3. CHEMICAL SHWTS The general empirical principles governing the chemical shifts of carbon atoms in amino-acids and small peptides are quite well established’22*23’ and the theory is advancing rapidly. No new theory is likely to be needed to explain the shifts in larger molecules, although progress is needed in the understanding of solvent and related shifts. There will, in general, be severai minor contributions of this latter type to each “C shift in larger structured peptides and proteins, SO that at high resolution one may expect to find markedly separate resonances from atoms which, on

Carbon-13-NMR

of Peptides

and Proteins

TABLE 1. Carbon L-amino

of acid

ala Cu CD co arg CL7 Cl CY C6 Ci co asn Ca Cb CY co asp Ca CL? CY co asp (Cy protonated) :; CY co cys Ca Cl3 (Z;“, Ca CLJ co cys (deprotonated) CL? W co gln Ca CP CY C6 co glu Ca Cl3 CY C6 Co glu (C6 protonated) CZ CP cy C6 co

gly Ca

co

his (imidazolium) Ca

CP CY C62 Gl co his (imidazole) CU Cb CY C62 Ckl co ile Ca Cb C.j’l CY2

Peptide shift range in DrO 50.8-51.0 17.5-17.8 175.8 54.6-54.7 28.8-29.0 25.3-25.8 41.7 157.5-157.6 175.0-175.2 51.3-51.8 37.2-37.8 175.4-175.9 172.9-173.3 52.5-52.7 39.5-39.8 178.4 174.2

Denatured shift range

protein in DrO

50.9-51.5 17.5-18.0 -

48.0 18.2 171.8 51.7 29.5 24.7 40.3 -

54.0-54.3 29.CL29.3 25.6 41.7-41.8 157.4 -

171.0 49.3 37.1 171.2 170.3 -

51.4-51.6 36.9-37.4 39.5 -

51.1 36.5 175.2 173.7 55.8 27.6 171.7 52.3-53.0* 38.9-42.8* 168.9-171.7*

51.4-51.6 36.4 -

49.2 36.3 ‘171.5 170.3 -

58.4 27.1 173.6 54.9-55.1 26.9-27.6 32.1-32.3 178.7-178.9 174.2-l 74.6 54.9-55.1 28.2-28.9 34.3-34.6 182.3-182.8 174.8-175.3

-

-

54.1 26.9 31.0 177.9 174.6 43.2-43.6 172.8 51.9 27.1 134.9 118.9 129.4-129.5 170.9 52.9 29.3 137.2 118.4 133.9 172.5 59.6-61.1 36.8-37.0 25.4-25.6 15.7-16.3

51.6 40.3 169.7

51.4 27.6 30.0 173.5 170.9 -

27.3-27.8 31.5-32.0 27.7-27.8 34.6-34.8 -

51.4 27.6 30.0 173.9 170.9 42.1 168.7

43.7-44.0 _.

136.7 59.5-59.7 37.4-37.7 25.4-26.3 15.8-16.3

c~--_cBH2-cyl,

0

\

CH-CH2-YO/a B

s

\

cH-%b-,H

/‘x

s \

CH-CP-SH

C'"

ICH--cgH2--S-1, /” f

‘CH-Cj&-C.-COpHz

/‘” s

\

CH-CjH2-C$i2”~O-

-

27.6-27.8 32.2-32.5 -

-

\

c’

C’” 0

-

134.1 -

Side-chain structure

Peptide shift in dimethyl sulphoxide

-

1

51.8 27.6 133.6 116.8 130.3 56.2 37.1 24.2 15.1

\

CH-CH.-CH ,/Q p

b

-“oOH Y’

OLIVER W. HOWARTH and DAVID M. J. LILLEY

6

TABLE 1 (Continued)

Carbon of t.-amino acid ile CS co leu Ca CD CY CSl C62 co lys(-NH;)Ca CP CY Cl5 CE co lys(-NH,)Ca CP CY C6 CE co met Ca CS Cv ck co orn Ca

Peptide shift range in D20 11.3-12.5 174.8 53.6-54.0 40.5-41.0 25.2-25.7 23.1-23.5 21.6-22.2 175.6 54.4-54.9 31.0-31.8 22.8-23.1 27.1-27.5 40.3-40.5 175.6 55.0 31.6 23.2 32.3 41.3 -

Denatured protein shift range in D,O 11.5-11.9 53.6-53.7 41.1-41.3 25.0-25.6 23.c23.4 21.6-21.9 54.5-54.8 31.1-31.5 23.0-23.4 27.0-27.6 40.5-40.6 -

11.0 170.6 50.5-50.9* 39.6*-41.0 24.0-25.2* 23.&23.7* 21.6 170.8*-171.7 51.8 31.5 22.1 26.6 38.6 171.2

51.5 32.0 29.3 14.6 170.9 -

57 37.1-37.6 136.7-137.5 129.5-129.9 129.1-129.5 128.2 -

pro(cis)Ca CD CY C6 co

61.0-61.3 32.8-33.1 22.9-23.2 48.4-48.8 175.8

-

58.7 31.7 22.0 46.8 -

ser Ca

56.6-56.7 62.1-62.3 172.6-173.2

-

CP co

54.8 61.6 169.7

thr Ca C/3 cy co

60.0-60.2 67.9-68.3 19.6-20.0 172.7-173.3

60.0-60.2 68.0-68.3 19.6-20.0

58.0 66.6 19.7 169.9

trp(pH 2-4)Ca

56.2 28.8 109.6-109.9 125.3-125.4 127.5-127.7 136.9-137.2 118.9-l 19.2 112.6-l 12.9 118.2-120.3

55.9-56.0 -

53.1 27.9 109.7-l 10.1 123.5-123.8 127.2 135.8-135.9 117.8 111.0 118.0-118.2

2CY Ci co pro(trans)Ca CS CY Cl5 co

Cl3 CY C61 C62 GX2 ck3 Cl2 Ci.3

61.9-62.4 -

109.3-109.9 124.9125.3 118.5-118.9 109.3-109.6 119.8-120.0

CH,U

CH-CH -CH ,B’ ;\ CH$ C’” 0

\ C~~-~-C~2-C.2-C.-NH; 0

\

-

15.2 -

/

\

-

53.9 31.0 30.2 15.0 175.0 54.6 29.3 23.5 39.7 173.5 56.5-56.8 36.637.1 137.2-137.6 130.3-130.4 130.1 128.5 176 61.6-61.8 30.2-30.6 25.3-25.9 48.U9.1 175.8

CF CY C6 co phe Ca CP CY 2cs

Side-chain structure

Peptide shift in dimethyl sulphoxide

CH-C /a P

-CH -CH -CcH2-NH, 92 62

s

c)-cp-c.y2-s-c~, 0

53.6 36.9*-37.7 135.9*-137.8 127.9 129.0 126.1 170.2*-170.9 59.1-61.1* 29.0-30.3* 24.1-24.2 45.8-47.6* 169.0

B

6

C/c\NFH~CH \ lY2 Cl+-_CBH+

2” 0

\

C$-CjMzOH

c’ 0

Carbon-13-NMR

of Peptides and Proteins

7

TABLE l.(Continued)

Carbon of L-amino acid trpCq2

co tyr Ca CL? CY 2C6 2c.e

Cl

co w CY 2C6 2ck CC val Ca

CP

CVl c;2 co

Peptide shift range in DzO

Denatured protein

shift range in D20

122.5-122.9 -

121.3121.6 -

56.2-56.6 37.337.6 128.7-129.4 131.4-131.7 116.8 155.6-155.8 176 123.1 131.6 120.1 166.1 60.7 30.8 19.3 18.5 174.8

56.8-57.0 31.1-31.6 130.9-131.2 116.0-116.2 155.5 -

Peptide shift in dimethyl sulphoxide

Side-chain structure

120.5 -

36.9 127.9 130.1 114%116.5*

\ c,”CH-CH ..q2

6

c Otl

’

122.5 166.5 60.5-61.0 30.8-31.1 19.6-20.5 18.8-19.3 -

the basis of primary structure alone, one would expect to be magnetically almost equivalent. Careful studies(2”29) have shown that all the 13C resonances of an amino acid unit, more than two units away from a chain end, in an unstructured polypeptide, are effectively independent of their neighbours. This important conclusion is modified only when the neighbour is proline; it means that, with only these two exceptions, any chemical shift differences between similar carbons in the same amino acid at different positions in a protein must be due to secondary or tertiary structure and not to primary structure. The latter is, in any case, relatively easy to find nowadays by classical means, so that “C-NMR holds great promise for the unambiguous study of the former, under conditions with which X-ray crystallography is not possible. The actual shifts observed for the amino acid X in the linear peptides NH,gly-gly-X-gly-gly and TFAgly-gly-X-ala-OMe, together with other shifts obtained inter alia from oxytocin,‘30-33) gramicidin’34.35’ and luteinizing hormone-releasing hormone(36) are listed in Table 1. Only the first two and the last are unstructured; the others, marked *, are not highly structured, and are included for comparison. There have been many similar but less definitive studies’37-52’ leading to almost the same shifts, although some earlier studies quote shifts relative to rather exotic, solvent dependent resonances (e.g. acetate ion, methyl iodide) which often differ from those in Table 1 by a constant factor of up to 1Sppm. In addition, authors do not always specify whether their references are internal or external,.although this can alter a quoted value by 1 ppm for some solvents. The shifts in Table 1 are adjusted to make them relative to internal tetramethylsilane, TMS: for aqueous solutions they will be closely similar relative to exter-

57.0-58.0* 30.8-32.4* 19.0-21.2* 17.0-21.2* -

\CH_C~CH3Y c’ 1 0

“CH

3Y

nal TMS. The table also includes shifts derived from the spectra of denatured hen egg-white and human lysozyme (pH 1.5-2.0) and bovine and human lactalbumin (pH 8.0-8.5) denatured in 8 M urea using 0.1 M 2-mercapto ethanol to reduce disulphide bonds. These were measured at 67.9 MHz, and assignments were aided by amplitude comparisons within the pairs of closely-related proteins. (53) The shifts of the corresponding zwitterionic amino acids are also very similar when allowance is made for the presence of the peptide bonds.(42.44,48,54,55) For residues preceding proline, C, shifts by an average 2.0 ppm upfield, and C, by 0.8 ppm upfieid.‘56) These shifts are comparable to those found upon alkylation of aliphatic amines.(22’ 3.1. Factors that AfSect Chemical Shifts 3.1.1. pH. Table 1 contains alternative shifts for the six amino acids, asp, cys, glu, his, lys and tyr, that are commonly encountered in either of two forms within the normal biochemical pH range. The relevant pK, values are respectively (asp) 4.0, (cys) 9.2, (glu) 4.4, (his) 6.7, (1~s) 10.5 and (tyr) 9.8, although these may be substantially modified in proteins. The peptide bond itself is effectively independent of solution pH. Two theoretical explanations of the pH shifts have been advanced. One assumes the domination of electric-field shiftsc5” and the other considers only the effect of protonation upon mean electronic excitation energy and charge density.@8~59) The latter theory gives good results in the cases with which it has been tested. However, the former theory, while consistent with much protonation data, does not explain the rather small shifts (typically c2 ppm) observed when similar molecules, such as EDTAf6” and CYDTA@” are bound to metal ions with charges varying between

8

OLIVER W. HOWARTH and DAVID M. J. LILLEY

1 $ and 4 -t . Such small shifts are more likely to be due to through-space shielding. Hence, although some perturbations may be expected, simple ion-binding is not likely to be reliably monitored by changes in 13C shifts alone. The same considerations imply that i3C shifts in structured proteins are likely to be relatively insensitive to changes in the ionic state of nearby residues, unless these are actually H-bonded to the residue under study, or else unless the pH change also induces a conformational readjustment. 3.12. Thro~h-sake shieldi~. In principle, the proximity of any other atom to a given 13C atom can shift the resonance either upfield, via diamagnetic shielding, or downfield, via temperature-independent paramagnetism.~62~ Each contribution is substantial (k 100-300 ppm), but in practice the two almost cancel, with the difference being hard to estimate, even in sign. Nevertheless, some progress is currently being made in the explanation of shifts, which arise from such subtle effects as different rotamer populations!63*64i The shift difference usually observed between the two leucine and valine methyl carbons illustrates this (Table 1). The diamagnetic and paramagnetic contributions are least likely to cancel when the diamagnetic shielding of the nearby atoms is highly anisotropic, as is the case with aromatic rings and with carboxylate groups and indeed peptide bonds. The resulting through-space shielding have been fairly extensively studied in 1H-NMR,(65) and should be very similar in i3C-NMR.‘66) They should, therefore, contribute about +2 ppm to the chemical shifts of nearby groups, with the sign of the shift indicating the relative orientation of the aromatic rings. A downfield shift, i.e. increased 6, will imply that the atom is approximately in the plane of the ring, and an upfield shift that it is approximately above or below that plane. It should be noted, however, that conformational changes may be as important as “ring current” shieldings, as has been shown in cyclic dipeptides.‘67’ Severe steric compression can cause upfield shifts of as much as 5 ppm.‘22’ 3.1.3. Solvent. Solvent effects in ‘%-NMR are comparable in size to ring-current shifts. A change of solvent may affect a shift either by the above shie1ding m~hanism, or by altering the charge distribution of the solute, or by inducing a ~nfo~ational change(6*+69) typically by making or breaking H-bonds. Only the first two mechanisms are relevant to the dimethylsulphoxide (DMSO) versus water shifts in Table 1. In general, the shifts in DMSO are slightiy upfield of those in water, with the effect on polar regions (Co, C, C-N) being greater, about 2-4 ppm. The corresponding shifts in methanol, where mea.sured(27) are about 1 ppm upfield, except for the carbonyls (about 2-3 ppm downfield). As the foundation for an important series of papers(7~70-7at Urry and Mitchell and co-workers have studied the various tetra-, penta- and hexapeptides derived from the

repeating unit of the fibrous protein elastin. and have shown that the solvent shifts of the carbonyls between DMSO and either water, methanol or trifluoroethanol are approximately halved when that carbonyl is H-bonded. A similar observation has been established for ‘H-NMR (NH protons) and both methods may be combined in principle to identify the internal H-bonds in a peptide. The extension of such studies to the whole protein is discussed under protein structure. The method was also shown to work for oxytotin and gramicidin-S, insofar as the 13C- and ‘H-NMR measurements correlated; it gave a credible H-bonding model. Small solvent effects ( +O.l ppmj have even been observed on going from H,O to DzO. These are mainly associated with labile proton binding sites. In the case of a terminal carboxylate, the i3C shift can be as large as +0.34ppm, which offers another ~signment possibility~*‘.“~ 3.1.4. Cis-trans ~so~erjs~ in proline. The most useful early apphcation of 13C-NMR in the study of peptides was the clear distinction it permitted between cis- and trans-proline. The resonances of C0 and C, each shift by 2-3 ppm as the form alters (Table 1). An example of such isomerism is shown in Fig. 3; there are many further examples. (16,26,36,39.42.46.51,52.78-96)

The

pans-form

is thought

to be stabilised by hydrogen-bonding in some cases. and also by steric constraints. Thus in N-acetyl DL proline”” the pK, values are 3.47 (cis) and 4.13 (tram). A solvent change from DzO to DMSO-d, or to pyridine d, also reduces the percentage of cisproline,(g4’ with H-bonding being implicated in the terminal proline.‘*es2’ It was at one time claimed that all proline-containing peptides longer than about six units contained only frans-proline. Certainly small peptides, both linear and cyclic, often show substantial or even total cis-configuration(9*.g9) whereas upon chain-lengthening (notably in Bovey’s study on oxytocin)(33) the proportion of &-configuration drops sharply. However, a high-field study of the octapeptide pro3-pro5-angiotensin II showed three minor cisconformers in addition to 85-9Oo/0 of a trans-conformer.“@ Furthermore, helix-coil studies of poly t-proline”OO*lO’) showed a substantial proportion of cis-prolines. The AG of cis-truns conversion of N-acetyl DL proline 1971was shown to be 3.8 kJ mol-‘. A similar study in chloroform using other blocking groups gave AGs from 0.6 to 1.3 kJ mol-I. AG$, the activation energy, was shown to vary from 72.3 to 80.2 kf mol-‘.~gy’ 3.1.5. Other identz~a~ie ca~f~r~~ltio~a~ changes. The conformation of a cystine S-S bridge relative to a diketopiperaxine (cyclic dipeptide) ring may be estimated from circular dichroism studies and also monitored by changes in the 13C shifts!10Z*‘03)Similar conformational changes have also been invoked to explain the shifts attendant upon the final ring-closure of oxytocin!30*37i Shifts attributable to second-

Carbon-13-NMR

of Peptides and Proteins

Complexation

of K+ to the antibiotics valinoand nonactin,” “) for example, shifts the six ester carbonyls downfield by 4ppm, and also has a smaller but significant effect on the other neighbouring carbons. These shifts are explicable in terms of a conformational change which makes the antibiotic into a better chelate. In the case of valinomycin, the .~nfo~ationai change is also reflected in T, changes.“is’ An additional measurement that this study made possible was that of AH for the K+ ion exchange, which was detectable via line-broadening. Attempts have been made to explain the ion-binding shifts of several peptides in terms of an electric field shift caused by the cation. When (val-pro-proala-phe), was complexed to Na+, two equivalent carbonyls were deshielded by over 2 ppm, whereas the other four were less affected.” 16v1“) Beauvericin, a symmetrical trimer of N-methylphenylalanine and a-hydroxy valeric acid (the -0 analogue of valine), showed similar d~hieldin~ upon complexation to both Na* and (slightly Iess) K+ (116). Valinomycin, however, shows smaller shifts with Na+ than with K+, Rb+ or Cs+, the latter being closely comparable.“‘*’ An alternative explanation of the ion-binding shifts which is more consistent with the inorganic data(60*61) is that upon compiexation certain carbonyls become deprotonated, either by loss of an internal H-bond or by desolvation. This deprotonation may be less complete with a smaller metal ion in a large, cryptate-like cage. A recent X-ray crystallographic study”‘9’ confirms that in at Ieast two uncomplexed conformers of valinomycin, four of the six ester carbonyls are involved in /I-turn-like H-bonds, which are broken upon complexation to Kt. Also studies of crown ethers confirm that NaC often has a lower co-ordination number than K+ ?t20) That ion-binding can produce substantial shifts in quite distant carbons is confirmed by Liinas et at. in a recent study of ferrichrome peptides.‘121) When these bind to A13+ (mainly via N-hydroxy N-acyl ornithine sidechains) shifts of +2 ppm are observed at distant a-carbons; for example, the three glycine C, values, which all appear at 43.6 ppm in the uncomplexed cyclic hexapeptide, shift to 41.9, 43.7 and 45.2ppm upon complexation. The ornithine fl and y carbons also shift upfield by an average of 2 ppm, as might be expected if they became sterically compressed against other atoms upon complexation. The a-carbon shifts parallel observed proton shifts, and may well be relayed to changes in H-bonding as the conformation of the peptide alters. 3.1.7. Paramugnetic s/t@. The binding of a paramagnetic shift reagent to a specific, definable site leads to substantial and often quantitatively meaningful shifts of most nearby ‘H and “C resonances.“’ Bayer and Beyer (tz2) find (on the basis of a limited study) that Pr 3+ binds specifically to the C-terminal carboxylate in oligopeptides; this can assist in nondestructive sequencing. However, Kessler and ~oltert123) observe that Eu(fod), interacts quite non~y~inll”~llS>

II

IllI I70

I

I

I

I

I

I

I

130

IllI

II 40

0

PPm

Proton noise-decoupled ‘“C-NMR spectra of solutions in DMSO-d, of two linear prolinesontaining peptides. T = 40”. (a) H-L-Thr-L-Phe-L-Pro-OH(b) H-L-Phe-LHis-L-Thr-L-Phe-L-Pro-OH. The letters c and t identify the resonances of the j?- and y-ring carbons of cis and zruus proline, respectively. The solvent resonance is at 39.8 ppm. FIG. 3. Investigation of proline cis-trans equilibrium (reproduced from FEBS Letterd8”).

ary structure A (104)

have also been described

in gramicidin

Many workers have investigated the helix-coil transition in polypeptides. The resulting structural shifts must be treated with care, because of the unusual solvent systems often chosen.dos-“o) Often the observed shifts (e.g. Co and C, downfield 3-4ppm on coiling) are explicable on this basis alone. The few experiments in aqueous solvents(6E~1’1J are discussed below under protein chemical shifts; they give shifts of about l-2ppm do~fie~d upon coiling for Co, C, and C,. For this reason it seems likely that helix-coil shifts will be mainly of use in the study of non-aqueous peptides; structured parts of alamethicin dissolved in chloroform/methanol have been identified in this way.“r2) 3.16. lun ~~~~i~g. From the previous d~s~ss~on it was apparent that the binding of a diamagnetic ion per se is unlikely to produce dramatic shifts, even when the ion is highly charged. However, large shifts (* 10 ppm) can arise for bound carboxylate acid carbons if they become deprotonated on binding, and corresponding but smaller shifts (- 1-3 ppm) will. occur in neighbouring carbon atoms. These shifts may be simply regarded as pH shifts (Table 1) induced by the effective pK, changes associated with ion-binding. In addition, there may well be substantial shifts if ion-binding alters the molecular confo~ation~1’3)

9

10

OLIVER

W. HOWARTH and DAVIDM. J. LILLEV

specifically with protected dipeptides (in non-aqueous solvents). Blout and co-workers(124’ have studied the binding of Yb(fod)3 and Eu(fod), to proline-containing cyclic. dipeptides, and show that in these compounds the remaining side-chain has several comparably populated configurations if it is aliphatic. However, if it is aromatic it markedly prefers the conformation in which the aromatic ring lies over the diketopiperazine ring. (124’ Further applications of paramagnetic relaxation agents are discussed in the section on the assignment of single carbon resonances in proteins.

was endo-puckered, i.e. with C; on the same side of the C,-C,j-N-C, plane as C,,. In contrast. free proline has 1.5 Hz as the corresponding coupling which fits a ring-puckering conformation averaged between endo- and e.yo-, or alternativeiy pianar.‘92’ A study using ‘J,, is described in the iater section on selective enrichment.

5. SPECTRUMASSIGNMENT

IN

PEPTIDES

peptides containing less than about seven amino acid residues are generally easy to assign by their shifts alone, even when some of the residues are 4 COUPLING CONSTANTS abno~al~i38*139’ In fact, 13C-NMR is quite a useful 13C-‘H couplings are relatively easy to detect in way of identifying abnormal residues and resolving amino acids and very small peptides.~36~42~‘25-‘28) unseparated mixtures, particularly when there are no 13C-13C couplings can also be detected in structural shifts to complicate the spectrum. However, there may be assignment problems when ‘3C-enriched amino acids,‘i2” and 13C-15N couplings in ‘$N enriched peptides. (130’ The latter (3J,-N) seem an amino acid is repeated in the oligopeptide, or when two conformations are simultaneously present unlikely to yield useful side-chain structural informaand only slowly interconverting. A careful study of tiond3” although some hope remains for the determination of the $ angle in the main chain.(i31’ The peak areas as a function of solvent, temperature or pH is necessary to identify the latter phenomenon;‘s3’ former lead to very complex spectra when the enrichment is non-specific and fairly high.” 32-134’ ‘Jcc is such a study may also yield thermodynamic parnormally 30-60 Hz and, although pH dependent, does ameters. Ambiguities arising from repeat units may often be resolved either by’a pH study (the terminal not depend in any obvious way upon conformation. 2Jcc is smaller than 3Jcc, often unmeasurably SO. 3J~~ residues will show an extra pK,) or by a solvent study’72’ revealing H-bonds. A further variation on can be 3.5-7.5 Hz between C, and Cd in an appropriate amino acid, but between C,, and C, it is less the solvent study is to exchange H20 for D20, decoupling only the protons. This can identify cr-carbons than half this value and is therefore usually undetectable. An exception is Co-C, coupling of 3.5 & 0.5 Hz by indicating that they are bound to a terminal -ND2 (1:2:3:2:1 multiplet), a peptide -ND (1:l:l multiplet) in cyclu gly-pro,@” which is somewhat conformationally restrained. Partial conformational restraint or or tertiary N (a singlet). Small H/D solvent shifts are else an aiternative coupling pathway aiso makes the also observed,“40’ as are reductions in T,. Where the C,-C, coupling in free proline undetectably small; the proton resonances can be identified, e.g. from their temperature or solvent dependence, then 13C greater restraint in gly-pro, cycle gly-pro and gly-progly raises this to a detectable 3.8 + 0.5 Hz.“‘*~~‘. resonances can be assigned by selective decoop3JCHbetween rw-carbons and b-protons could in ling.‘86’ Another assignment method is to use selective and principle yield side-chain conformations if observable identi~able chemical modi~cation. Methods of doing in seiectively~nrich~ proteins.“26*i27’ Bystrov and co-workers(13s.136’ have given a recent assessment of this are steadily being developed for peptides and especially for native proteins; they are discussed in a the prospects for this. Only two “biochemical” applications of three-bond coupling constants have so far later section. If all these approaches fail, then the last resort is been reported. One is in a substrate binding study. Rodgers and Roberts(‘37’ used ‘Jcx between the ace- to construct a 13C or 2H’75’ labelled peptide. As an tyl carbonyl carbon and the a-carbon in N-acetyl example, this has been done for all ten amino-acid units in gramicidin A, where the labelled biosynthesis tryptophan to investigate the binding of this substrate to &chymotrypsin. Using a somewhat extended extrais fairly simple.“41’ For peptides with more than polation, they deduced that 3J,-- changed from about six units, such labelling may well be necessary +2.3 +O.lHz to -0.8 4 0.8 Hz upon binding. for a secure assignment, as Griffin et ~1,‘~” showed Hence, using a Karplus relationship, they deduced the in a reassignment of the well-studied nonapeptide likely bound conformation. The angle 0 was found oxytocin following a selective enrichment at gly-9. to be nearly 90” which gave a meaningful binding The detailed assignment of resonances in proteins model. is only just beginning, and will require the combined The other is of 3Jcc in thyrotropin releasing factor use of most of the methods developed for peptides, (TRF) in which the proiine residue was enriched in together with the use of known structures, where 13C. The Co-C, coupling was found to be the most available, to estimate plc perturbations and throughdefinitive for determining the conformation. The space shieldings of nearby groups. Some detailed coupling of ~0.5 Hz showed that the pyrrolidine ring examples are discussed in a later section.

Carbon-l 3-NMR of Peptides and Proteins

5

I&

I-

‘%’- N" - t0 -C"-NH-:! 04gt

0 -

in

11

130

CH 133 88 -Cl+-WI,-CONH,

002-t N!M!l weot

nT1 values in ms observed in oxytocin in deuterated dimethylsulphoxide. are nT, values obtained in aqueous medium (see ref. 14).

Numbers in parentheses

Primary sequence of luteinizing hormone-releasing hormone showing nT, values obtained at 25.16MHz for carbons bearing protons in D20 at pH 6.4. rtT, values are in ms; n is the number of directly attached proions. Sample con~ntratio~ 100 mg/ml. Temperature, 32’. T, values determine using 20 spectra 10,000 scans/spectrum, T = 2.0s, r = 10-250ms. Superscripts indicate overlapping resonances. FIG. 4. Some typical peptide T, values. (Fig. 4a reprinted with permission from Biochemistry. Copyright by the American Chemical Society(r4” ) (Fig. 4b reprinted with permission from Biochem. Biophys. Rex C~~~~~~l 5zt).

6. RELAXATION TIMES IN PEPTIDES

Fuller details of the use of the spin-lattice relaxation time T, and the spin-spin relaxation time T2 are given in the section on proteins. For almost all peptides the molecular tumbling rates r, fall in the region which makes

where n is the number of chemically attached protons,
will be much less, but can yield very useful structural information concerning the distances between the carbon and the least distant protons.‘142) Peptide chemists have been interested mainly in T, values for 13C nuclei as an aid in structure assignment. Because chemical shift studies frequently proved to be ambiguous or inconclusive, it was hoped that motional analysis would reveal local conformational restraints. Results have been obtained for cyclic dipeptides,@” tripeptides,‘g3) linear di- and tri-peptides(‘43.‘44) related linear pentapeptides@4*26) and higher peptides such as alamethicin,‘” 12) lutenising hormone releasing hormone (LHRH),“45,146) metencephalin~147’ tetragastrin~‘48‘149) angiotensin,” 50) valinomycin,“‘4’ oxytocin,os’31 “I gramicidin-S,053)

-

. .

_ _

poly(OH.pro-gly) poly(gly gly pro gly) poly(pro) poly(OH.pro) poly(y-benzyl L-glutamate)

pWvo-gb)

Polypeptides :

gramicidin A valinomycin valinomycin-K + LHRH met-encephalin

gramicidin A

.

_,

Peptides: H,N+ gly gly X gly gly COOH H,N+ gly gly X gly gly COOHIN gly gly X gly gly COO(1Ys)s cycle@-trp), oxytocin oxytocin lysine vasopressin (ile-5) angiotensin II (prospro’) angiotensin (pro3pro5) angiotensin gramicidin S

Compound

0.36 0.36 0.26 0.61 0.68 1

5” 0.19 0.15 0.34.4 0.3

15

0.1 l-O.30 0.130.53 0.1 l-o.28 0.16 0.24 0.5 0.88 0.5 0.5 0.3 0.2 0.33

refr or r&s

water water water water water CDCl, + CF,COOH

50/50 DMSO/DzO

D2O

DMSO DMSO DMSO

CD,OD

CD,OD

Dz0

DzO

D2O

DMSO DzO DMSO DzO

DzO Dz0 Dz0 Dz0

Sokentb

TABLE2. Correlation times (reTIor r&s)

-

5.5 6.8

-

3.5 3.5 4.5 4.1 1.1

l-3 neutral 9-11 7.5

PH/PD

40

32 32 32 32 32 40

50 50 35 38

32 -> 32 32 25 40 > 43

33.5 33.5 33.5

T/“C

Comments’

Random coil (some aggregation?) n-helix. MW 70

200 mg/ml

Less at lower pH, 100 mg/ml Some T control problems 67.8 MHz, 67 mg/ml 150 mg/ml; intermolecular aggregation? Helical dimer; aggregation at 100 mg/ml?

Solvent viscosity ratio 1.9: 1

Some aggregation?

of some peptides and proteins

.

109

179 179 179 loo loo 109

115 115 145, 146 147

104

102

2426 24-26 24-26 111, I62 67 151 152 151 150 16 16 153

Reference

78

w: -15

20

D2O

H20

D2O

native native

D2’3

H2O

H2O

H2O

water

6.5

7.4

4.7

3 1.5 6.5 5.5 6.9

3

neutral

7.0

37

35 32 23 10

42 28-32 36 36 36

42

40

10

45 45 30 2 12 12 31 34 30

22

T/C

Native. MW 14,000 but slight aggregation tendency Native, 13.8 mM. MW 14,000 but slight aggregation tendency No cone”. dependence MW 18,000, 8.6 mM MW 12,000, 16m~ MW 65,000 (tetramer) 3.7 mhi MW 6000,but 50m~ From carbonyl Tr values Estimated from linewidths MW 6000; estimated from tyrosine linewidths “rs” = 35 ns at 6.35 Td

MW 12,000. 5s same via Raman scattering Almost random coil Native. .MW = 14,000 Random coil. 36 residues a-helical trimer (Helical portion) MW 14,000 (Helical portion) MW 56,000 Random coil Assumes rigid His side-chain Approx. value from enriched linewidth. Hexamer? Assumes rigid/enriched His

Comments’

“T,, measured from a linewidth, implies T, = 1 ns. A large error in the quoted Tt could be possible because it is almost on the T, minimum. ’ Aqueous solvent usually includes buffer and salt. ’ Molecular masses rounded to nearest thousand. d The evident discrepancy in ‘TV measured at different fields indicates the limitations of the theory on which 7R is calculated. Current research by one of us (OWH) indicates that when internal librational motions are included, the discrepancy is removed, and lower values of

bovine serum albumin

basic pancreatic trypsin inhibitor ligamentum nuchae e&tin fibrous a-elastin ~~~sf~idja ferredoxins

22 22 * 5 17-19 17 47

20

lysozyme

lysozyme various myoglobins horse myoglobin horse-heart cytochrome-c human adult haemoglobin

‘D20

22 H20

D2O

27

H,0,‘200/, J&O

H2O

H2O

H,0/300/, f&O H20/300/, Da0

H2O

Hz0

D20

1.4 6.55 4.8 4.8 neutral neutral 1.0 range range

24 rf: 3 0.45 110 -20 -45 0.5 18 0.3

DzO

PH/PD

0.85 f 0.10

Solventh

neutral

7&s

DzO

or

I I-14

7eff

tryptophan synthetase cf subunit lysozyme

Proteins: muscle Ca’+-binding parvalbumin ribonuclease-A ribonucle~-A collagen peptide al-C,2 collagen peptide al-($2 histone histone tetramer (H3-H4), calf-skin gelatin a-lytic protease (I -gly) insulin

Compound

TABLE2 (Continued)

7R

are generally indicated

171

188, 189 190 7 191

21 166, 187 21 21 21

21

11

186

181 181 56 56 182 I83 38 184 185

176, 180

Reference

14

OLIVER W. HOWARTH and DAVID M. J. LILLEY

lysine vasopressmL5’) and the (rather different) gramicidin-A.(‘04’ These are extensively reviewed in reference (6) and two representative examples are given in Fig. 4. Sufficient studies have now taken place to permit the following useful if tentative, generalisations: (a) In all T1 studies, and particularly in those of non-protonated carbons, great care must be taken to exclude paramagnetic ions. For example, order of magnitude errors were made in the carboxylate T, values of free aqueous amino acids because of iron(II1) impurities in commercial Dz0.054X155’ Chelating agents such as EDTA are not always adequate to remove such ions, particularly at low pH. Similar cautions have recently been voiced following a i5N-NMR study.05@ (b) In free amino-acids, small peptides and free terminal units of longer peptides, the side-chains are hardly more mobile than the a-carbons. Even methyl groups do not show nT, values more than three times the lowest value. In contrast, the methyl groups on the non-terminal acid side-chains of larger peptides usually have T1 values close to nine times as long as the a-carbons, as predicted by Doddrell, Glushko and Allerhand for a rotationally unrestricted methyl group attached to a relatively immobile molecule.“57’ Later studies”58-160) have shown that this behaviour is to be expected for all carbons more than four bonds away from the backbone, provided that each internal rotational diffusion coefficient is greater than the overall diffusion constant for the molecule. This explains the original generalisation, because overall diffusion is easier than internal motion for a small molecule, but not for a large one. (c) Enhanced side-chain motion is sufficiently normal in peptides of more than about seven units for its absence to indicate steric hindrance.‘15’) Lysine, for example, shows an average TI increase along the side-chain of x 1.6 per carbon (slightly less at the main-chain end, slightly more at the -NH: end). This is approximately as expected from studies on linear hydrocarbons. (161) A corresponding factor of x 1.7 has also been observed in poly-lysine.“62’ The charged terminal group does not seem to reduce the motion in water significantly by creating an ionic hydration shell; indeed, it is almost always remarkably free in aqueous proteins, despite proposed lysine saltbridges. The side-chain of arginine is somewhat more restricted in motion by the mass of the guanidinium group. Even aromatic groups often show T1 evidence of rotation relative to the main chain,t’50*‘53’ although this is not always apparent.‘14s*‘s1) Such motion lengthens the T1 values of the o- and m-carbons, but because of the rigidity of the ring leaves that of the p-carbon comparable to that of the B-carbon. This, of course, implies that the rotation is primarily about the C,&;. bond. (d) If the main-chain motion of a larger peptide is hydrocarbon-like and hence unrestricted, then the

terminal r-carbon should have an ttT, of about 2-3 times that of the central r-carbons. Factors of this size are observed in linear pentapeptides”“‘“’ but the factor is nearer 1.8 in LHRH and 1.3 in angiotensin. The latter is almost certain, therefore, to be constrained by specific interactions involving the terminal groups. Rings will often be more constrained than chains; in the case of oxytocin (I5 ‘) the constraint is more than can be explained by simple dynamics. The implied steric hindrance may be related to the relative resistance of the ring to enzymatic cleavage by chymotrypsin. Some estimated values of rcrr are listed along with protein values in Table 2. Glycine commonly shows a motional enhancement of at least 20% over other r-carbons. This is presumably a simple dynamic effect due to the absence of a side-chain. Proline is almost as restricted at C, as at C,, although C, and C;. are 5O-100% more mobile. (e) A detailed study of smaller peptides in DMSO@‘) suggests that peptides with more than two polar areas (e.g. peptide bonds, charged terminal groups) tend to carry a layer of solvent with them as they rotate. T, values in DMSO are about half those in water, as might be expected from the relative viscosities in the absence of major hydrophobic effects. Lowering the pH can significantly increase T, values (see Fig. 4), presumably by reducing the average amount of non-specific, time-averaged H-bonding in the peptide. Solvent composition can also affect aggregation and hence T,.” “’ (f) Nuclear Overhauser enhancements (NOE) of carbons bonded to hydrogen are usually at a maximum (9 = 2.0) in peptides, as is expected when the Larmor frequency is much less than rR. NOE values of quaternary and carbonyl carbons typically give q = 1.1, indicating a substantial dipolar contribution, which will increase if the carbon is reasonably near to other protons!2’,‘42,‘63’ Such NOE values will also change dramatically on change of solvent (e.g. D20 ---, H20) as the latter provides most of the nearest protons.‘*l’ (g) Ion-binding will in general affect tumbling correlation times, although the changes will not be large unless the molecular shape changes dramatically. For example, on binding of K+ to valinomycin(1*5’ r1 changed from 0.19 to 0.15 ns (CDjOD; WC) indicating a more globular conformation for the complex. A similar but larger change was observed with the antibiotic nonactin.“63’ Unfortunately, the effects of conformation upon T1 for a molecule of constant volume are not particularly larget15’ although they may be detected by very careful measurement.“46) Conclusions relating to peptide dynamics should be equally applicable to substantial segments of random-coil peptide where these occur in proteins. 1. PROTEINS Three main features separate protein spectra from peptide spectra. Resonances of structured proteins are

Carbon-13-NMR

of Peptides and Proteins

15

FIG. 5. Theoretical log-log variation of nT,, nnT2)-* and NOE with tR for an isotropically tumbling molecule whose 13C relaxation is dominated by dipolar coupling to n directly attached protons. The curves for unprotonated carbons are similar except that the vertical axis is shifted and the longer T, values reduced by spin-rotational and (at high field) CSA ~nt~but~ons (redrawn from J. Chem. PhysoJ7)). Field values in kG. generally broader. The shift scatter of given aminoacid carbons is also usualiy greater in proteins, although it is generally masked as an especially broad line in spectra recorded at below 45 MHz. The signalto-noise ratio is commonly poor because of the broad lines. An understanding of protein 13C spectra therefore requires some underst~ding of how Tr and Tz vary with moiecular motion and spectrometer frequency. Further details may be found in references (157) and (164). 7.1. relaxation

Times, T, and T2, in Proteins

The main features of the relaxation theory of macromolecules were laid down some years ago by Doddrell, Glushko and Al~erhand~15’) They have since been confirmed experimentally insofar as the overall protein tumbling times ‘tR which they predict are closely consistent with values obtained by other methods. For a carbon attached ta a single. proton, under conditions of complete proton decoupling, the (exponential) relaxations are given by the equations l/T, = l/l0 ~CS(%i - wc) + 3f(%) + 6f(yl + Ml l,T, = l/20 AC4rc I-f(OH - WC) + 3f(o,) + 6f(ww) + 60% + ~YXI

where A s f?ytfy$Z,< rj’ 6> (symbols as before) and f(w) = 7,& + m27&; zefr being the effective rotational correlation time for that C-H bond. Although T1 and T2 are slightly altered in the presence of anisotropic rotation, they remain equal in all cases under the “extreme narrowing” or “white spectrum” case that (o+, + o.&C,lf < 1. These equations are illustrated graphically in Fig. 5, which also shows how the resulting NOE varies from q = 2 to the minimum vahre of q = 0.1. The loss of NOE and ~inewidth at high field when (yr + w,)z,~~x 1 is apparent. The effect of side-chain motions has been incorporatedo5” in a further simplified model in which a tetrahedral CH, group with rotational correlation time zG is attached to a rigid, otherwise motionally symmetric& molecule of correlation time tR. Figure 6a shows a plot of nT, versus 7R at a field of 1.41 T for various values of zG, and Fig. 6b the corresponding plot for nT& The NOE drops off with increasing 7G much as for the isotropic case (Fig. 5) provided that 7R is relatively large (> 10e8 s). The same is also true for Tr except that in the range fO-“o c rs < 2 x 10e6, Tr is anything up to ten times less than its high 7R value in Fig. 5. In other words, for a given relatively rapid sidechain motion, T1 and T;’ can be decreased substan-

OLIVERW. HOWARTHand DAVID M. J. LILLEY

16

(a)

IO

(b)

0.1

FIG. 6. Theoretical log-log variation of (a) nT, and (b) (nrrT,)- ’ with TV for various 5,; conditions as in Fig. 5 except that the relevant carbon is permitted one degree of internal rotational freedom, about the C,-C, bond. No allowance made for anisotropic tumbling or for cross correlation effects (redrawn from J. Chem. Phy~“~s’)). 1.41 T.

tially by protein motions within the range normally encountered. This paper also proves the important result that when ro 6 ta, T1 is exactly nine times that observable in the absence of independent side-chain motion. This is a useful test for pinpointing the relative values of ~c and ~a. At the other extreme, carbons on unrestricted sidechains which- are further than four atoms from the main chain will have T, values almost independent of Q. It is immediately apparent from Fig. 5 that the measurement of a TI value in itself leaves an ambiguity in 7R. Thus in Fig. 5, an nT, of 60ms at 2.35 T could result from a ta of either 1.2 ns or 15 ns. A decision between such values may not be obvious, and can lead to substantial dispute.(165) One way of resolving the ambiguity is to repeat the TI measurement at a different field, and then to fit the various T, values to graphs such as in Fig. 5. This has been done for myoglobin at 1.41 T and 2.35T,(16@ but is clearly not a convenient procedure. Another possibility is to measure the NOE. A third possibility is to measure T2, which is not bimodal. In many cases an inspection of linewidths will suffice to identify the true value of ta. However,

linewidths are not easy to measure accurately, and may well be dominated in native proteins by the overlapping of resonances. A possible solution to this, which does not require special apparatus, is to measure T2 by a repetitive Carr-Purcell type A pulse (90” - 7 - 180” - 5 - FID - long sequence, i.e. delay-),. This has been briefly mentioned by Miziorko and Mildvan(167) without details. However, errors in such measurements could arise either from incomplete proton decoupling, or from exchange of energy between the proton and carbon spins during the 2~ time interval.“68) An empirical study(169’ shows that the former objection is not serious and that the latter process, whilst creating large errors for ~a 2 10-r’ s, only leads to small errors at more normal protein 7R values (< 10e9 s). The resulting spectrum simplification may also be useful, as may the ready visual-distinction between lines broadened by relaxation and lines broadened by overlap. The simultaneous measurement of T,, T2 and NOE could, by a variation on the above argument, yield some side-chain bond angles.(r7” Allerhand et al. have recently(171) extended their calculations to include elliptical protein shapes. They

Carbon-13-NMR

of Peptides and Proteins

find that the resulting additional field-dependence of T, is relatively small, so that overall T1 measurements are not promising as a means of measuring molecular asphericity. They also note that the observed field dependence of the T, values of 13C nuclei in bovine serum albumin and of lysozyme is anomalous, although that of myoglobin and haemoglobin is not. It is noteworthy that the two anomalous proteins are known to self-aggregate; this could radically affect their motions if the aggregation were relatively specific. 7.1.1. Factors affecting7R. Table 2 lists a wide variety of effective rotational correlation times of pep tides and rotational correlation times of proteins, from which the following generalisations are apparent : (a) 5,ff for the central cc-carbons of a random coil molecule is not strongly dependent upon chain length beyond about six units. For example, 'leffincreases only by a factor of 3 between (lys), and (lys),. As a comparison, n-alkanes show similar behaviour. The value of 7,ff between n-decane via n-hexadecane to polyethylene (30°C) changes from 0.011 through 0.04(161) to 0.070.~172~The corresponding value for polypropylene”“) is 0.24, which shows the constraining effect of side-chains. Comparable results have been obtained with other polymers,“73’ although further side-chain steric constraints increase 7.ffsomewhat more. In this context, it may be noted that increasing proline content increases 7effin the prolinecontaining polypeptides. Proline may have been implicated as a structure-initiator in RNase folding. (174*17s1The longer 7,ffvalues are probably comparable to 7R.'153) (b) The formation of secondary structure, whether or not tertiary structure is also formed, results in an approximately hundred-fold increase in 7ef,. In at least one case the resulting 7eff has been shown to equal 7R within experimental error, the latter having been measured independently by a Raman depolarised light-scattering method.‘176J Other equations of this kind have also been made, although with less strict comparability. (‘w It seems that, as expected, most cc-carbons in folded parts of proteins are effectively immobilised. (The same is certainly not true of methyl groups, nor of some side-chains and may not be true of glycine a-carbons.)

(c) Globular

17

proteins

of molecular

acid-denatured ribonuclease A crs-oxidised ribonuclease A various native myoglobins partially denatured myoglobins random-coil gelatin bovine serum albumin” basic pancreatic trypsin inhibitor

be a consequence of its near-spherical ellipsoidal shape (300 x 360pm) and the surprisingly long value for the triple-helical collagen peptide, even for 2”C, may be evidence for its being an elongated ellipse, possibly further slowed by some random-coil sections at the ends of the helix. The measured 7R is probably determined mainly by the end-over-end rotation because of the long-axis orientation of the C,-H bonds.“@ However, aggregation is also possible as a slowing mechanism.““) The effect of structure-formation is particularly evident in the comparison of the cyclic decapeptide gramicidin S and the biologically but not structurallyrelated dimeric pentadecapeptide gramicidin A, both in methanol. (d) For zR > lOns, even at 1.4T, the NOE enhancement should be very close to q = 0.1. This has been confirmed experimentally.(166) (e) Estimates of (anisotropic) 'Lo values are beginning to appear from measurements on solid or semi-’ solid samples. Fibrous cr-elastin has a linewidth of approximately 100 Hz for the glycine C, resonance,(‘) with similar or smaller widths for other resonances. This indicates a motion in which static ‘dipolar coupling is almost averaged out, and in which 'sefc is around 35 ns. Also rapid anisotropic motion has been identified in the collagen fibrils of (cross-linked) calf achilles-tendon and rat-tail tendon.““) In this latter study a special high-power decoupling technique was necessary. Further studies were made of reconstituted fibrils of chick calvaria collagen, enriched at either C, or Co of glycine. The T2 (linewidth) showed a large (700 Hz) residual static relative main-chain rigidity coupling, indicating (7R > 10-3 s) along the fibril axis. However, T1 and the NOE were also measured for both enriched atoms, and were 0.1 s and q = 0.35 for C, and 1.55 and q = 0.6 for C,,. These are comparable to the values expected for a “rigid” protein in solution with 7R - 20 ns. It remains to be seen whether this motion merely involves the (previously noted) extra mobility

C,

C;

Cd

(0.36) 0.25 0.27

(0.23) 0.19 0.18

0.12 0.15 0.20 0.46-0.70 0.18

J estimated from linewidth-25”C, pH 5.3. h See Table 2 for experimental details.

,.P.\.\I.R.S.

I2 I-B

around

lar radius, which implies an attached hydration shell. The small 7R for muscle calcium-binding protein may

TABLE 3. Lysine T,~~values (in n$

native ribonuclease A

mass

14,000 daltons all seem to have a zR between 20 and 25 ns in water. Such T,, values correspond to hydrodynamic radii slightly larger than the actual ,molecu-

0.20

CC 0.070-0.08 0.076 0.09 0.18-0.22 0.13 0.08 20 0.10

OLIVER W. HOWARTH

18

of glycine residues in peptides, or whether it represents a general main-chain mobility in collagen fibrils. 7.12. Factors affecting 5,. The inspection of almost any carbon NMR spectrum of a folded protein shows the presence of side-chain motions, particularly for C, and C, of lysine but also often for methyl carbons. Because these resonances are narrow, and often have a substantial NOE, they tend to dominate the spectrum. It is of considerable interest to know whether a side-chain is effectively rigid within a globular protein (this might well imply a structural role) or whether it has extra motional freedom. Little is as yet known about the relative freedoms of sidechains on the interior and the exterior of proteins; if some general principles could be established for this then a carbon NMR spectrum would yield further useful information about tertiary structure. This section describes the information currently available for some types of side-chain. Lysine. In random-coil poly-lysine, the following values of reff have been observed”62) in I&O solution (ns). HN’

0.52 0.2

0.12

0.07

0.04

\ CH-CH~-CH~-CH,-CH2--NH: OC

/

Although these values are short, even the C, value, corresponding to a Ti of 0.6 s, still indicates constraint refative to the prediction for a truly unrestricted side-chain cr, - 2 s). The same holds true for the penultimate residue in lysine vasopressin, even though the terminal residue is only glycine (amide).(lsl) When (1~s)~undergoes a random coil--, helix transition around pH 11, the rcff values all lengthen dramatically, to an extent which may imply aggregation prior to precipitation. The relative narrowness of the lysine C, and C, resonances in native proteins was noted in the earfy published spectra of ribonuclease-A,‘r9*’ lysozyme,“) carboxymyoglobin ‘and haemoglobin.” g3) Table 3 give some reported r,rf values (in ns), TABLE 4. rG

DAVID M. J. LILLEY

These results should be treated with some caution. In many cases they refer to overlapping peaks from quite different resonances. Inevitably they are a weighted average of the various individual carbon relaxation times in the molecule. This average will be dominated by the most mobile residues because of their narrowness and full NOB. That even the C, resonance in lys is not invariably narrow is apparent from any spectrum of native bovine serum albumin. The bracketed C, and C, figures in native RNase A (Table 3) must surely represent a mobile minority of lysine residues, as otherwise impossibly rapid rotational diffusion about the C,--C, bond would be implied. Methyl groups. It is normal for a side-chain methyl group to have an nT1 value of two to four times the next carbon, because there is little to restrict its rotation.(22*‘58) In medium sized peptides, where the methyl group is comparatively free but the next carbon’s rotation is hindered by the bulk of two methyls (vat, leu) the nT, enhancement is more typically five to eight fold,‘14s*i50+iJi) with actual T, values of 0.2-0.5 s. Application of the simple model described earlier(157’ gives an approximate ho s lo-l2 s; an infinitely small to would only give a nine-fold enhancement on this model. Some methyl so values (ns) estimated on the above basis, are given in Table 4. The corresponding T, values (ms) are included in brackets. References and details as Table 2. The protein to values observed are all somewhat greater than in alkanes (0.01-0.03),‘16L~ indicating some residual motional restriction of the methyl groups. However, they are all much less than the folded protein rR values in Table 2. It follows from the T, values that individual methyl resonances, even in very slowly rotating pr&eins, should have linewidths of 20-40 Hz.~‘~‘) These values will be further reduced by reduced protein rR or by the use of higher magnetic fields. In fact, Jones, Rothgeb and Gurd have observed individual methyl resonances in “C enriched methionine in native sag-whaIe myoglobin whose linewidth is about 5 Hz-barely more than the internal dioxan standard.(lg4) It is of interest to note that two peaks were observed, separated by 1.4 ppm, for Met 55 and 131. This confirms the impli-

values for methyl groups in polypeptides and proteins (in ns) methionine

gramicidin-A dimer native myoglobins partially denatured myoglobin collagen peptide trimer random-coil gelatin acid-denatured ribonuclease-A cvs-oxidised ribonucleaae-A native ribonuclease-A “,z+aii”,nrss(bawd on simple theory).

1

and

0.005 (200)

,

leucine <0.002” (250-280)b 0.07 (72-83) < 0.002 (280) 0.04 (100) valine + threonine 0.005(204) 0.003 (247) v 0.02 (148)

valine co.003 (250)

alanine 0.004 0.06 0.004 0.04 0.03

(220) (84) (211) (100) (120)

0.004 (221) 0.004 (218) -0.03 jllli)

-

Carbon-13-NMR

Carbon

0

of Peptides and Proteins

- 13 Magnetic

19

resonance

&I al

( 0)

e $0

a &I 5

u

4

: :

g

.E c

$

a

Aortic

a-

elastin

(23%)

So Iution

I

( b)

Aortic

Q

elastin

(48C)

Coacervah

L

1

200

160

160

149

I

L

120

100

I

00

60

40

20

wm FIG. 7. 13C spectrum of fibrous aortic a-elastin compared with simplified analogues (reproduced from Biochem. Biophys. Res. Commun.“‘).

of Fig. 1 that the main contribution to the apparent methyl linewidths in the spectrum of a native protein is chemical shift scatter. Similarly narrow resonances were observed for both methionine and lys (NME;) enriched at the methyl carbons and incorporated in cytochrome-c. The narrowness persisted even in spectra from the intact N. crassa cell in which the labelled biosynthesis was carried out; (the observed chemical shifts showed that no free enriched methionine precursor remained).og5) An even more dramatic demonstration that protein methyl groups rotate rapidly comes from a spectrum of fibrous aortic elastin, essentially a solid sample

cation

(Fig. 7). The chemical shift scatter in this sample is drastically reduced because of the five-unit repeat structure of elastin. The two valine methyls have linewidths of only about 20 Hz, and are thus clearly resolved. A weaker ala C, resonance can also be identified. Jones, Rothgeb and Gurd have also used their S-methyl enriched myoglobin for the first reported temperature dependence of the T1 value of the 13C in a native protein. They obtain an activation energy for the methyl group motion of about 12kJ mol-’ for both Met 55 and 131. This activation energy agrees well with an earlier estimate based on the exchange rates of internal protons.og4)

peptide

A

trimer”

dimer”

ribonuclease

ribonuclease

inhibitor

168 c0.u

350’ [0.091d

CO.061

109-127

86114 [0.08] 162 [O.lO]

460 [0.03]

glu, gin CY

Experimental

I06 [rigid]

CO.51

c;z

179

CY

leu

T, values

“Substantial uncertainty in rc owing to the insensitvity of T, to ro. b Calculated by authors from NOE of q = 0.4, for asp, phe, leu /I. ‘T, in ms. d ho in ns (based on simple theory).

trypsin

t-glutamate

helical poly y-benzyl

basic pancreatic

parvalbumin

gelatin

random-coil

A

A

muscle Ca’+-binding

myoglobins

denatured

native myoglobins”

gramicidin-A

cys-oxidised

acid-denatured

native ribonuclease

collagen

LHRH”

oxytocin

5.

C6

w

TABLE

[ l&20]b

65 [rigid]

CS

val

and estimated

70 [rigid]

Cl1

200

trp CP

[TJS

4&50 [rigid]

Cl1

180

C62

his

for protonated

40-50 [rigid]

ck

his

side-chain

E

80 [rigid]

80 [rigid]

CO.31

CO.~l

4&56 [rigid] 101-107 co.351

Ill 100

213 co.071 231

80 [rigid]

10.31

111

Cl1

Cl1

57

160-200

138 CO.41 230

132 CO.41 210

trp CH arom

tyr Cc

tyr Cd

lo.:] [O.E]r;;

C6,

phe

carbons

.r

d

?

21

Carbon-13-NMR of Peptides and Proteins It would appear that the potential information obtainable from the methyl portion of protein spectra, and possibly from other aliphatic side-chain motions, has been underestimated to date. However, two cautions should be sounded about the estimation of methyl group ro values from Ti values. For all amino acids except alanine the to model described above is not strictly applicable, even for isotropically tumbling proteins. Furthermore, when alanine methyl Ti values have been studied in detail(‘sO’ including a measurement of NOE, they fit badly to the model. Ti appears to be too long for the observed NOE. A possible explanation of this is that non-exponential T, relaxation may be occurring. This has already been observed in the methyl proton NMR of tetragastrino4’) and in the “C-NMR of methylmercuric species. (ig6) It arises from the effect of cross correlation terms in the relaxation matrix of the methyl group, and is likely to be important for alanine C, when to is fifty or more times larger than TV. Other protonated side-chain carbons. Values of nTi (in ms) for various other reasonably resolved and unambiguous side-chain resonances are given below, in Table 5, with estimated approximate rG values (in ns) in brackets, calculated according to the approximate model of references (21) and (157). The possible ambiguities in 7G were resolved by an inspection of linewidths, supported in the case of myoglobin by related NOE values (21) and in the case of ribonuclease by T2 measurements.“6Q) The Table includes some peptides for comparison. It is immediately apparent from the Table that a measurement of Ti is frequently a poor measure of TV and can be quite ambiguous. The value for glu and gln C, in native myoglobin is a case in point. The Ti value alone supports either a rigidly bound side-chain or a ~c of 0.1 ns. The latter value is tempt-

ingly similar to the denatured value. Furthermore, this resonance appears to be fairly narrow in the spectrum. However, the longer 7c value offered in the Table might be selected by analogy with the other proteins. It is also possible that both values are simultaneously valid, with the narrow component of the resonance being attributable to some relatively unrestricted side-chains. Evidence for such microheterogeneity of oo values is given in the next section. In helical poly y-benzyl L-glutamate the shorter 7G is chosen unequivocally on the basis of linewidth and NOE.‘lOg’ One generally-agreed conclusion seems to be that aromatic side-chains in native proteins possess relatively little motional freedom. Even in peptides they commonly have ro values larger than or little different from rR. In addition to the observations of Table 5, rigid or almost rigid histidine has been observed in a-lytic protease”a4) and tryptophan synthetase.“*6*iQ7) Also only slow rotations of aromatic rings have been observed in the ‘H-NMR of lysozyme.‘1g8’ A possible exception to this principle is that phenyl rings often appear to be non-rigid, possibly due to rocking motions.080) More mobility is commonly observed for substrates and 13C labels, as discussed in a later section. However, in many cases the increase in mobility is not such as to indicate a very short 7(;.‘It was also found necessary to include some mobility beyond C, in the aliphatic side-chains of the structured parts of various histones in order to explain the observed spectra.‘182’ It is apparent that further study of side-chain motions is much needed. More attention should be paid to the estimation of T2, which varies monotonically with 7G. Typical linewidths for a methylene carbon should increase from 12 to 70Hz between ro = 0.05 and 200 ns at 1.4 T.““) NOE measure-

TABLE6. Calculated and experimental T1 values (s) of non-protonated aromatic carbons and C: of arginine residues of native proteins at 1.42 T Carbon” Tyr C: (H@) Tyr CF (DAY Phe C: Trp C,, (HAY Trp C,, (DzO) His C: (H,O) His C: (HzO)“ His C, (D,O) Tyr C;. Trp G2 Trp C, Arg C: (HzO) Arg Cc (DsO)

Cytochrome-c Calculated Exp.b

Lysozyme Exp.b Calculated

0.45 0.79 0.40 0.57 (0.73)1 0.94 (1.50) 0.36 (0.40) 0.45 (0.52) 0.46 (0.53) 0.40 1.09 0.51 0.21 (0.27) 0.87 (- 5)

0.49 0.87 0.44 0.62 (0.80) 1.03 (1.65) 0.39 (0.44) 0.49 (0.57) 0.51 (0.59) 0.44 1.20 0.56 0.23 (0.29) 0.94 (4 6)

0.4 0.4 0.3 0.4 0.7 0.4 0.4 0.7 0.4 0.2 0.4

0.5 0.6 0s 0.7 0.3 0.4’ 0.4 . 0.8 0.5 0.40.7 0.9-1.5

Haemoglobin calculated 0.97 1.72 0.86 1.22 (1.59) 2.02 (3.27) 0.77 (0.86) 0.98 (1.13) 1.00 (1.16) 0.86 2.36 1.11 0.46 (0.58) 1.84 (-11)

ilSolvents are given in parentheses for carbons that have theoretical T, values that change significantly when going from HZ0 to DZO. b Experimental value. c Histidine residue in the imidazolium form. d Histidine residue in the imidazole form. eHistidine residue in the imidazolium or imidazole form. ’ Calculated values in parentheses omit dipolar contribution from l“N.

OLIVERW. HOWART~and DAVID M.J. LILLEY

22

ments should also give information. However, the dependence of NOE enhancement n upon ro is complex, being low (r] - 0.2) at both large and small ro for the as values typically encountered in proteins, but high (rl - 1.4 at 1.4 T) for intermediate to ( - 0.5) values. One further piece of evidence concerning side-chain motions comes from a magnetic susceptibility study of the helix-coil transition of poly-y-benzyl-L-glutamate in a liquid-crystalline phase. The aromatic sidechains appear to acquire substantial motional freedom in a preliminary transition prior to the actual uncoiling transition.“ggr Unprotonated side-chain carbons. In several ways the study of the motions of unprotonated side-chain carbons is harder than that of protonated carbons. The linewidths, whilst resolvably narrow, tend to be dominated by inhomogeneity rather than by Tz. Also the Ti values are comparatively long, often inconveniently so, and those of exterior residues may be affected by paramagnetic impurities. The study of these resonances is dominated by the work of Oldfield, Norton and Allerhand on lysozyme, cytochrome-c, myoglobin and haemoglobin.” ‘) The relevant resonances are arg C, (guanidino), his C,, phe C,, trp C,, C,, and C, (bridgeheads), and tyr C, and C, (phenoiic). Their conclusions may be summarised as follows: (i) The aromatic carbons all have a dipolar-coupiing dominated relaxation, and their NOES (a = 00-0.3, +0.2) indicate an effective absence of internal motion. The actual T, values for cytochrome-c and lysozyme are reproduced in Table 6, where they are compared with the theoretical values calculated from the known rR and interatomic distances, and the assumption that to = co. The authors noted some small differences between the same carbon on individual residues, and also some significant increases in Ti on going from HZ0 to DzO (i.e. from NH, to ND, in arg, his, trp). (ii) Dipolar coupling from directly-bonded i4N is

shown to be of importance for his CT, trp C, and arg C,. The bracketed calculated values in Table 6 are those which ignore this interaction: they are all too long. (iii) The arginine C, resonances in HEW lysozyme have Ti and NOE values that betray both internal motion and intramolecular heterogeneity. At least four separate resonances can be resolved for the 11 arginine residues in HEW lysozyme. The three most: prominent have Ti values of 0.12, 0.54 and 0.43 s in HzO, and 1.47, 1.27 and 0.90s in D30, which are all appreciably longer than would be expected for rigid side-chains. Also the mean NOE is PJ= 0.5. In contrast, if paramagnetic effects can indeed be excluded, then the corresponding residues in horse heart cyanoferricytochrome-c appear to be rigid. One word of caution needs to be given concerning the T, values of non-protonated carbon resonances. NOE measurements on r3C-enriched urea can be carried out in which either the solvent (H,O) proton resonance or the NH1 resonance is irradiated independently. The former makes the greater contribution to the NOE and hence presumably to the .T1.(200) This observation is probably also relevant to arginine, and thus may help to explain the surprising rigidity of these residues in cytochrome-c. It may also open up another means of distinguishing internal residues from external ones, in addition to the use of relaxing agents.@ ‘) One or two other relaxation times have been reported for arg C, in random-coil peptides, but they are generally comparable to the recycle time of the T, measurement, and hence unreliable. Even the longer values quoted above are open to this objection.

7.2. Structural Shifts and Assignment of Resonances 7.2.1. Fitting to peptide shijts. Until recently all published 13C spectra of native proteins were obtained at fields no higher than 2.3T. The natural linewidth at this field for protonated carbons is equiv-

0

Gx

ST 0 +-NN(~N-HIw0> x k -

asp

his

(

.

.

ser

FIG.8. Proposed scheme for the catalytic activity of a-lytic proteasc (reprinted with permission from Biochemistry.‘*84’ Copyright by the American Chemical Society).

Carbon-13-NMR

alent to OS-3 ppm depending on the degree of internal motion. Thus, although the presence of line broadening via chemical shift scatter is apparent (particularly from an experiment which measures the true T2, or from resonances such as tyr Cc, which should be very sharp) the actual resolution of the scatter was unlikely. Indeed, many spectra showed only broad envelopes for the a-carbons and for the p-carbons. Nevertheless, many of the better spectra of native proteins do show resonances attributable to only one or two types of carbon atom, and thus some reasonably convincing assignments have been made by comparison with known amino acid and peptide ~~~f~~~7~56~69~166~181~182~187~193~201~204~

No

major

de_

viations from peptide shifts have been recorded. However, the methyl carbon region has generally been difficult to assign, presumably because of substantial structural shifts. As a test case one may consider the Cd resonance of isoleucine, which is 4ppm upfield of all other resonances and hence unambiguously assignable. The peptide shift is 11.3-12.5 ppm (the lower value referring to gly-gly-ile-gly-gly). Observed values for its mean shift in native proteins range from 9.2 ppm (basic pancreatic trypsin inhibitor”sg) through 10.0 (histone H4) to 12.9 (ribonuclease-A).“s’) It is possible that other such “structural shifts” may have been missed in the past because of the assignment method. 1.2.2. Selective enrichment. The most unambiguous assignment method is via selective 13C enrichment. We have already noted the structural shifts of up to +2.0 ppm observed for methionine in sperm-whale myoglobin.“g4,205) Hunkapiller et al.(184) have made a detailed study of the single histidine residue of a-lytic protease, enriched at C,. This is a serine protease, having a “catalytic triad” of aspartate-histidineserine in a “charge relay” system. They were able to follow the ‘Jc- and chemical shift as a function of pH. Over the range 5-9, the chemical shift titrated between 134.5 and 137.Oppm, in response to a single ionisation with a pK, = 6.7. This pK, has been frequently observed for the activity of serine proteases, assumed to be following the ionisation of the active histidine imidazole group. However, Hunkapiller et ~l.“*~~showed that the i.Jcn remains typical for neuTABLE 7.

23

of Peptides and Proteins

tral imidazole over this range (by comparison with model compounds). Below pH 4 the histidine resonance became split, with three slowly interconverting forms being present, corresponding to neutral and protonated species. The shift with pK, = 6.7 must therefore reflect the titration of a nearby group, assumed to be the aspartate of the triad. Thus they showed that within the active cleft of the enzyme, the normal pK, values of aspartate and histidine were reversed an interesting example of local major perturbations within hydrophobic clefts. This reversal makes good “bio-organic sense”, since the catalytic triad (Fig. 8) becomes electrically neutral in the transition state, thus obviating the need for the charge separation required in alternative schemes. Ti, T2 and NOE measurements showed that above pH 3.3 the histidine is held rigidly within the protein but below this pH one of the three resonances observed corresponds to considerably greater mobility. In another study, the four histidine C, resonances in similarly enriched tryptophan synthetase a sub-unit were not separately resolved even upon deuteriationos6) (In fact, deuteriation does not always greatly reduce i3C linewidths unless scalar relaxation of the second kind is removed by strong 13C{2H) decoupling.“97)) However, the available practical resolution in this experiment was only l-2 ppm, so that a probable 2 ppm scatter of chemical shifts was merged into one broad resonance. Both histidine-12 Ci206) and also all phenylalanine- carbons have been enriched in the synthetic N-terminal 1-15 peptide of ribonuclease,‘207) and the shifts have been measured for the random-coil pep tide, the peptide bound (in a helical form) to ribonuclease-S (residues 21 to 124) and the latter complex plus 2’ CMP (2’-cytidine monophosphate): 2’ CMP is a competitive inhibitor of the enzyme. The shifts are shown below in Table 7, together with comparable values for enriched gl~-6.‘~‘*) The linewidths indicated some residual motion, particularly for the phe side-chain. The histidine titration curve was very similar to that of enriched a-lytic protease described aboveos4) but was interpreted more conventionally. The shifts upon coil-formation are not large; currently the only available aqueous

Shifts, observed on binding of RNase-S’1-‘5) peptide to RNase-S’ (in ppm)

Carbon deprotonated his G phe Ca Cfi CY C6 Cc Cl Co gty Ca Co

“Peptide value” (Table 1) 137.2 56.5-56.8 36.6-37.1 137.2-7.6 130.3-0.4 130.1 128.5 176 43.243.6 172.8

1

RNase-S Free peptide

Complex

Complex + 2’ CMP (excess)

137.0 55.8 37.9 137.3 130.2 129.8 128.2 174.1 43.6 172.4

138.3 56.5 37.4 137.6 130.4 129.9 128.4 175.4 44.3 174.7

56.5 37.4 138.0 130.5 130.2 128.9 175.8 -

.

24

OLIVER

W. HOWARTH and DAVIDM. J. LILLEY

comparisons are in poly-lysine where coiling changes C,, by +0.4 ppm, C, by + 1.7 ppm and C, by +0.8 ppm’162J09’ and with poly-L-glutamic acid (Co, C, both +2.3 ppm, C, - 0.8 ppm).@s’ Even in these two cases, and especially for C,, it is hard to separate the influence of the helix formation itself from the overall effects of the protonation changes which cause it. 7.2.3. Natural abundance single carbon resonances, including paramagnetic effects. The main work to date on identifying single carbon resonances has come from the study of carboxylate and unprotonated aromatic carbon resonances. The carboxylate region of a protein ’ 3C spectrum often shows sharp, pH-dependent resonances around 6 = 182-3 from glutamate C,, and generally has other resolvable resonances also. Maurer, Haar and Riitergans(*“) were able to resolve many carbonyl resonances in the small (MW 6000) protein basic pancreatic trypsin inhibitor (BPTI). From the pH dependence of the spectrum they identified gln-7 and -48, asp-3 and -50 and the (C-terminal) ala-58.‘2’u) The spectrum of BET1 resolved single carbon also reveals other resonances.“s**l*g) Using a high-field spectrometer, Shindo and Cohen@ 1‘) tentatively identified the carbonyl resonances of leu-129 (terminal) and asp-52 in hen egg-white lysozyme, as well as bound acetate. They used the additional technique of selective line broadening by Co(I1) to assist the assignment. Co(I1) is known to bind at the active site of lysozyme, and therefore the identification of the asp-52 resonance holds promise for future solution studies of the catalytic action of lysozyme. The ring carbons of tyrosine (including the protonated ones) are resolvable in the small clostridia ferredoxin proteins, and are individually shifted by their proximity to the nearer of two not quite equivalent paramagnetic Fe.& clusters. Packer and Sternlicht and co-workers have made a detailed study of Clostridium pasteurianum and Clostridium acidi-urici ferredoxins, and have also obtained data on Peptococcus aerogenes ferredoxin.~*i2**i3) These are important agents of electron transport; in principle, each Fe&, cluster has five oxidation states, and in practice about three accessible ones. The tyrosine ring carbons shift by up to 18 ppm depending on the oxidation state of the cluster to which they are closest. The shifts are in either direction, and proton temperature variation studies(*14) show them to be due to both the paramagnetism and antiferromagnetism of the Fe4S4 cluster. The electron exchange between ferredoxin molecules is rapid (for reasons not yet fully understood) and this gives a smooth variation of shift with oxidation state rather than any splitting or exchange broadening. The assignment of the relevant aromatic resonances was achieved by techniques which are quite generally useful. In the apoprotein (Fe,S, clusters removed) both tyrosines have all four aromatic resonances

within 0.2 ppm of the expected value (Table 1). They shift in the ferredoxins and could be assigned via the partially or fully coupled spectra”” via linewidths and via peak areas. The distinction between tyr-2 and -30 was made by a chemical substitution of phenylalanine in position 2; this does not affect the structure significantly, and occurs naturally in C. pasteuriarwm ferredoxin. A further refinement in assigning this ferredoxin in the reduced state was the use of coherent off-resonance proton decoupling. This is of value when the relevant proton resonance frequencies are known; the attached carbons achieve maximum decoupling as the relevant proton frequency is traversed. The technique is illustrated in Fig. 9 in which the relative narrowness of the non-protonated carbons is also apparent. The actual analysis of the carbon shifts, once assigned, was dependent on previous proton work correlating the degree of oxidation with various shifts, and in particular that of the most downfield proton resonance. The final correlation of both the carbon (tyr) and the proton shifts with the degree of reduction is reproduced in Fig. 9. From these shifts the authors were able to deduce the relative midpoint redox potentials of each cluster pair. and also, by using a ferredoxin mixture, to find the relative potentials of the two Clostridia ferredoxins. They interpret the shift differences between these two ferredoxins as arising from conformational differences in the reduced rather than in the oxidised state. That the tyrosine residues are close to the Fe,& clusters is apparent from the quite large shift differences between the tyrosine carbons and the tyrosine protons. However, because of the electronic complexity of the Fe$, cluster, no detailed explanation of the absolute magnitudes of the shifts is yet available. The most detailed study of single carbon resonances so far published comes from a series of papers by Allerhand, Oldfield and co-workers, mainly concerning hen egg-white lysozyme” ‘.* i.’ 15--2’7’ and various cytochromes, myoglobins,~2’~2’6~2L8’haemoglobins’*‘*’ and P. aeruginosa azurin.‘218) The haem proteins, like the azurin and the ferredoxins. have variable oxidation states which can be helpful in assignments as well as being interesting in their own right. In summary, most of the predicted unprotonated single carbon resonances have been resolved-for example, 23 out of 28 in lysozyme(*“) and all 18 aromatic resonances in horse heart cyanoferricytochrome-c, and many assigned. The spread for a given carbon type is substantial even in the diamagnetic proteins; for example, a single six-fold degenerate trp C; resonance at 6 = 109.9 in denatured lysozyme becomes resonances at 6 = 108.8 ( x 2) 110.3, 110.8, 111.9 and 112.7 in the native protein. The detailed strategies for assignment are likely to set the pattern for many future protein studies. First, some attention had to be paid to maximising the spectrum quality. Optimum signal-to-noise was

Carbon-13-NMR

of Peptides and Proteins

25 % Reduction,

l4

,

0 ,

,

20 ,

,

40 ,

when X = I I

60 ,

80 I

loo I

1

Aliphotic Proton Shifts (ppm from most Oowfieldshifted

ppm

from

CS,

(a) Coherent and noise-decoupled r3C-NMR (lo”) spectra of the aromatic residues of fully reduced Chtridium pasteurianum ferredoxin. -40 mg/ml, pD 8.0. A, coherent decoupled. decoupling frequency is 9.0 ppm referenced to the proton resonance position of the sodium salt of 2,2dimethyl-2-silapentanesulphonic acid (DSS) (2.0 f 0.5 ppm downfield from 3’,5’-tyrosyl ring proton resonance). B, noisedecoupled. C. coherent decoupled. decoupling frequency is 7.0 ppm (0.5 f 0.5 ppm downfield from the 3’S’-tyrosyl ring proton resonance). Broken and unbroken lines connect tyrosyl and phenylalanyl r3C resonances, respectively.

achieved using 20mm sample tubes and appropriate crystal filtering. The relatively broad protonated carbon resonances were removed as shown in Fig. 3 of reference 21 to give convolution-difference spectra. Interestingly, the protonated resonances were sufficiently narrow to require additional broadening, via off-resonance noise-modulated decoupling, for their removal. The remaining unprotonated ready resonances were sufficiently narrow for further resolution enhancement(220’ to be generally unnecessary. An incomplete example of the resulting assignments is given in Fig. 10 which shows the aromatic and arginine Cr region of the convolution-difference spectra of horse heart ‘ferrocytochrome-c(A) and its ferri-(B) and cyanoferri-(C) derivatives. Assignments were made by the following methods: (i) Ojj+esonartce noise-modulated proton decoupling substantially broadens even the narrowest of protonated carbon resonances (this is generally valuable when a side-chain has rapid internal motion and hence no naturally broad resonances). (ii) Selective protorl decoupling at a frequency corresponding to 6,, _ 3 narrows arginine C: resonances (in D20 solution) but leaves tyrosine C: resonances broad. The broadening in tyr C: comes from the two

Position In Oxidized Protein)

(b) r3C shifts of the tyrosyl residues in reduced Clostridium and Clostridium pasreurianum ferredoxin as a function of the most downfield a!iphatic ‘H shift at different degrees of reduction. 13C shifts are relative to the corresponding ring carbon resonance positions in the oxidized protein at lo”. The most downfield ‘H shifts are relative to the most downfield aliphatic ‘H resonance position in the oxidized protein at E-17”. Arrows point to the ring carbon resonance of the Tyr’ residues of partially ( b ) and fully ( f ) reduced C. pasteurianum ferredoxins. To calculate the fractional degree of reduction of partially reduced ferredoxins from the ‘H NMR spectra (15) of aliquots of the r3C NMR samples, one subtracts 16ppm, the resonance position (relative to DSS) of the most downfield-shifted proton resonance in the oxidized protein, from the resonance position of the most downfield-shifted proton in the partially reduced protein. This difference is then divided by 40.4ppm, the difference between the most downfield resonance in the oxidized protein and the most downfield resonance position in dithionite-reduced ferredoxin.“’ This 40.4 ppm difference is assumed to represent 100% reduction. To calculate the fractional degree of reduction of partially reduced ferredoxins from r3C NMR spectra one subtracts the resonance position of the corresponding ring carbon resonance of Tyr’ in partially reduced ferredoxin and divides this by the corresponding ring carbon shift (relative to the oxidized protein) of the Tyr’ residue in fully reduced C. pasteurianum ferredoxin. acidi-urici

FIG. 9. 13C resonance assignment and analysis in terms of oxidation state, for clostridium ferredoxins (from reference (191)).

H, protons, and the decoupling at 6” 5 3 removes a cu. 8 Hz coupling from the two H, protons. D,O is necessary to eliminate proton coupling from the protonated nitrogens. (iii) Partially relaxed (PRFT) spectra can distinguish resonances with different relaxation times, notably trp C,, and C,, (see Table 6) from phe and his C;. Once again, D20 is necessary to enhance the T1 difference by deuteriating trp NJ. Conversely, use of HZ0 gives a second method for distinguishing arg C, (T, _ 0.2 s) from tyr CF (T, w 0.4 s). However, this technique relies upon the relevant carbons being immobile relative to the protein. (iv) A soluent change from Hz0 to D20 produces

OLIVER W. HOWARTH and DAVID M. J. LILLEY

26

0.1 ppm for trp C,, and C, and his C,, but of +0.13 ppm for tyr C; and trp C,, and +0.19 ppm for arg CF. These shifts are not sufficiently well established in proteins to be reliable assignment methods in themselves. However, they may provide confirmatory evidence, or even, as in HEW lysozyme, remove an accidental peak overlap. Some small shifts in his C, in the protonated form only may point to a new way of investigating the ionisation state of this residue. (v) pH oariation is of great value in the specific assignment of his C, resonances (it should work well for his C, also). The pK, values of histidine sidechains are often (at least partially) known from other work. For example, the two titratable histidines in shifts of less than

horse heart ferrocytochrome-c, his-26 and his-33, are the only resonances to show pH dependence in the range pH w 4-9. One resonance has a pK, of about

6, the other below 4.5. His-33 is known to have a pK, of about 6.5, and hence the other resonance must be his-26. Its low pK, makes the assignment in reference (184) less abnormal. A “normal” pH shift of one tyrosine C: resonance in the same protein implied tyr-103, as this residue is relatively exposed to the solvent. The N-terminal glycine carbonyl resonance was also distinguishable by its pH-dependence. (vi) Setecriue pm-magnetic relaxation by added ions, which has been widely used in ‘H NMR studies of proteins”) can also yield useful information in

TRP-59

Cy

Regions of aromatic carbons and C, of arginine residues in the convolutiondifference natural abundance “C Fourier transform NMR spectra of horse. heart cytochrome-c in Hz0 (0.1 M NaC110.05M phosphate buffer). Each spectrum was recorded at 15.18 MHz, with noise-modulated off-resonance proton decoupling, 8192 time domain addresses, a 4000 Hz spectral width, and a recycle time of 1.105 s. The convolution-difference procedure was carried out with 5 1 = 0.51 s, ~~ = 0.034 s, K = 0.9. Assignments are given in the text. A, 11.5mM ferrocytochrome c, pH 6.7, 40”. after 16.384 accumulations (5 h total time). Peak numbers are those of Table 2. B, 19.4mt.i ferricytochrome c, pH 6.9, 36”. after 32,768 accumulations (10 h total time). Peak numbers are those of Table 3. C. 19.4 mM cyanoferricytochrome c, pH 6.9, 36”. after 32,768 accumulations (10 h total time). Peak numbers are those of Table 4. The peak at about 114 ppm arises from excess free HCN which is in fast exchange with about 0.5% free CN-. FIG. 10.

Assignment of aromatic single-carbon resonances in the spectra of various cytochromes (from reference (21)).

Carbon-13-NMR TABLE 8. Assi~ments

Carbon

of single

Shift (ppm)

P

157.5-l 51.6 158.5 158.2 158.1 157.9 129.4-129.5 133.5-133.9 135.5 132.3 130.7 130.0 127.8 122.7 137.2-137.6 141.6 139.0 138.7 137.7 137.3 136.8 109.6109.9 113.3 112.7 111.9 110.8 110.3 110.3 108.8 127.5-127.7 129.6 128.5 128.5 127.8 127.8 127.1 126.7 136.9-137.2 138.9 138.3 138.0 137.8 137.7 136.4 128.7-129.4 134.0 130.7 130.7 129.6 129.0 128.3 127.8 , 126.7 155.6-155.8 157.9 156.8 156.6 156.5 155.7 154.8 154.5 153.5

L

“C i L

histidine C; ; C C A L ; pheny~alanine C; : L

tryptophan

C:

:: C C P A :: L

tryptophan

Cat

L P L L A : L

tryptophan

Cr2

k :: k L

tyrosine C;

tyrosine Ct

carbon r~onan~es cytochrome-c

Origin

arginine C;

4 A

21

of Peptides and Proteins in azurin,

lysosyme

and

Residue 79 u (C 38 or 91) u U

(protonated form) (imidazole form) 26 33 u 15 u 18 (coordinated to heme Fe”“) u (not 46) 3 or 38 3 or 38 34 u (2 carbons) 48 108 123 63 62 59 (2 carbons) 108 111 48 63 59 28 and 62 123 63 28 or 111 123 and (28 or 111) 59 62 108

?I? u

20 azd 53 u u n U

(2 carbons) (?103 for one) 20 or 53 20 or 53 u 2u3 67

A = P. ffe~uginosu azurin (di~a~etic form); L = hen egg-white Iysozyme; P = peptide value (from Table 1); C = horse heart f~o~tochrome-c; u = unassigned to any specific residue.

28

OLIVER W. HOWARTH and DAVID M. J. LILLEY

‘%Z-NMR. For example, Gd(II1) is a powerful relaxing agent which does not produce significant shifts, and is known from X-ray studies to bind near to the active site of lysozyme. Its relaxing effect should fall off isotropically as re6, where r is the relevant Gd-C distance. The resonances that were observed to broaden in this particular study were one trp Cd2, one trp C,, and one trp C,. These were therefore assigned to trp-108, whose rw6 value (C,) is more than five times that of the next nearest tryptophan C,. Further studies at higher Gd(II1) concentration have identified slightly more distant carbons.““) An important caution in such lanthanide binding experiments is to test the effect of binding (diamagnetic) La(II1) first. If this produces only minor shifts then one may assume that no significant conformational change occurs on ion-binding. The Gd(II1) is then introduced by successive replacement of La(II1). In the lysozyme study, the only substantial shift produced by La binding was in C, of his-15 (about 1 ppm upfield at pH 5). This residue is near to the binding site and the shift is probably a minor diamagnetic perturbation. Spin-labels may also be used as relaxation probes.‘* “’ (vii) Selective chemical modification may be useful in assignment provided that it does not affect structure. The site of modification can be identified by degradation studies. For example, the action of N-bromo succinimide (under mild reaction conditions) with HEW lysozyme exclusively converts the indole ring of trp-62 to an oxindole ring. This change should remove C,, shift C,, upfield by -6 ppm and leave C,, relatively unaffected. A new resonance should also appear for Cd,, around 182 ppm. These shifts were observed, although the assignment was complicated by the proximity of the indole of trp-63 and by splitting attributable to stereoisomerism of the oxindole ring. Iodination under various pH conditions is also selective and informative.““) (viii) Homologous proteins, provided that they have essentially the same tertiary structure, provide a natural equivalent of chemical modification. Thus a comparison of horse and sperm whale cyanoferrimyoglobins identified val-1 Co, and a tentative assignment of tyr-151 C, and C, and his-12 C, in the former; similarly a comparison of horse heart and Candida krusei ferricytochrome-c identified tyr-73 Cc of the latter, and a tentative assignment of tyr-52 C, and of his-32 C, and (as a pair) his-39 and his-45 C, in the latter. Further comparison with red kangaroo myoglobin “‘i’ has confirmed the assignments for C, and C, of tyr-151, and has also identified the corresponding resonances of tyr-146 and tyr-103. The latter were also shown by their pH-dependence to be in the protein interior. By a converse argument, if the peak positions of homologous proteins or subunits are closely comparable, then similar structures are almost certainly indicated. Thus a comparison of various haemoglobins showed considerable similarity (+ 1 ppm) in given trp shifts, even though

a 6ppm spread of shifts was noted in a given subunit.(‘l’ (ix) Diamagnetic ring-currenr shifts attributable to the haem macrocycle have an effect on nearby groups which may be approximately calculated. Thus his-18 of (diamagnetic) horse heart ferrocytochrome-c is actually co-ordinated to the haem iron. Its C:. shift of -7 ppm (upfield from the mean C.. position) compares with a calculated ring-current shift of -5 ppm. Similarly, tyr-67 C; shows a -3 to -4 ppm shift (talc. - 2 ppm). (x) Intrinsic paramagnetic shifts and broadening are immediately apparent in a comparison of the spectra of a low-spin ferro- and a ferri-cytochrome. In the latter paramagnetic species, all the haem resonances are broadened into undetectability, as is the CY of the histidine (his-15) co-ordinated to Fe(II1). The other resonances shift steadily as the ferro-form is slowly oxidised (the electron exchange is just rapid enough for all but one of the lines remain narrow throughout the oxidation). Thus the assignments of the ferro-species may be deduced from those of the ferri-species without further experiment. With the exception of tyr-67 (+ 10 ppm) the shifts are I 2 ppm. They can in principle be predicted if the structure is known. A similar study has been carried out with azurin.” i9) Table 8 shows the resulting assignments for horse heart ferrocytochrome-c and for HEW lysozyme, rearranged to emphasise the chemical shift heterogeneities. A detailed discussion of the origins of these has not yet been published. Inspection of Table 8 shows that the native protein shifts are more likely to be above the “peptide” shift than below it; this may be little more than a “solvation” effect. Trp-108 shows that upfield shifts for one carbon do not necessarily imply the same for another on the same ring; in fact the opposite might be expected from model studies!‘*) The general spread of shifts for a given carbon is around 4 ppm in the absence of paramagnetic shifts, although that for arg CI is considerably less (0.6 ppm). The shift of 4 ppm is more than is observed for non-specific solvent effects in small molecules, but less than that observed for strong interactions such as (partial) protonation. The small shifts of (hydrophilic) immobilised arg C; compared with (hydrophobic) phe, ile etc. argue in favour of ring-currents being the main cause of the more exceptional shifts. An intriguing possibility, which requires further investigation, is that substantial (> 1 ppm) structural shifts indicate tertiary rather than secondary structure. This seems to be supported by the large structural shifts observed for ribonuclease-A (Fig. 1) and the small shifts observed in the histones (Fig. 12) which have substantial secondary but probably little tertiary structure. The (reduced) heme shifts are also reported in this reference, and are further discussed via model compounds by Wiithrich and Baumann.(222-224)

Carbon-13-NMR

of Peptides and Proteins

7.3. Progress Towards Structure Determination in Proteins It is ekks~t Cram the previous section that the assigmmerir 6r S&e-c&an rSOnarrces ‘in _ur&6tns leans heavily upon previously determined X-ray crystallographic structures. The immediate biochemical usefulness of such studies lies mainly in the investigation d sma% per+mr~aS~onPz5~ w%ch hernor&Tab&9 leave most of the structure unaltered; ‘%-NMR can thus extend the validity of one crystal structure to homologous proteins and to protein complexes. Further development is likely to depend upon a reliable understanding of the detailed causes of structural shifts. Despite this, many of the assignment methods discussed above are potentially applicable to proteins of unknown structure. The use of mild solvent effects to identify exterior residues could be supplemented by weakly-binding relaxation agents such as Gd(‘r’) (EDTA)- or Mn(“) (CYDTA)2-‘60961) and by the measurement of side-chain T1 or T2 values. Specific, strongly binding paramagnetics~2’7~ can be used to “map” a protein structure, particularly if two wellseparated binding sites are available to permit “triangulation”. Further possibilities for the partial structure deter-’ minatian of proteins arise when the protein has some special feature, such as a repeat structure (elastin) or only partial random coil structure (histones). 7.3.1. Elastin. Elastin and collagen are the main protein components of connective tissue. Fibrous elastin is the insoluble, cross-linked core of elastic fibres; its unlinked precursor is called tropoelastin, and a partially linked, MW 70,000 fragment called a-elastin can also be prepared. Both proteins are filamentous and have many of the physical properties of fibrous e&tin, including an ability to absorb about 60:; by volume of water (as in the normal physialogical state). The sequence of tropoelastin has not yet been fully determined. However, although it is not totally regular, it does contain three closeIy related repeating sequences :

N

29

pro-gly-gly),-valOMe was shown to have unusual structural mobility in comparison to tropo- or a-elastin. The general extra mobility of glycine residues has already been nated,.and in addition the poiy-tetrapep: tihe may lack the seconci’d%ano: Iii contrast, the poly-pentapeptide HCO(val-pro-gly-val-gly),-valOMe has many close physical resemblances to a-elastin. Hence, when i3C-NMR solvent studies showed it to have the same fL6onds and hence the same basic (repeating) structure as the penta- and hexapeptides (Fig. ll), a comparable structure was indicated for a-elastin. To check this, the poly-penta-peptide solution was next compared with a solution of a-elastin at the same temperature.“’ The correlation is shown in Fig. 7. Although the cross-linked a-elastin gives a broader spectrum, with a few extra resonances, the chemical shift correlation in the carbonyl region is remarkable , and strongly supports the claim that the H-bonding pattern figure persists in dissolved rx-elastin. Finally, the close similarity of the spectrum of fibrous aortic a-elastin to the solution spectrum”’ suggests that the /?-turn structure is common to the native protein as well. The spectrum of fibrous aartic a-elastin is also remarkable for its relatively high resolution, which indicates substantial internal motions. This mobility is almost certainly related to its elasticity, as in rubbers. A ‘I,~~ of about 35 ns may be deduced from the linewidths, and one of 40 ns has been calculated from T, measurements on the carbaxylate resonances of calf ligamenturn nuchae (75% elastin by dry weight).‘lgO) In contrast, the highly cross-linked protein collagen is much more immobilised in its native state.(227’ 7.3.2. The histones. The histanes are a group of highly basic proteins of fundamental importance in maintaining chromasamal structure!228) Recent results suggest that a complex of eight histone molecules associates with a 200 base pair length of DNA to generate a bead-like structure.(228) Single histone

val-pro-gly-gly val-pro-gly-val-gly C i ala-pro-gly-val-gly-val 1

The first stage in the study of elastin was to synthesise and study the three peptides corresponding to these repeating sequences,‘70-72*75’ and also the cyclic per,~~~~~7 l’ 1% ~LVerQ> mutaZ& effects on the carbonyls, combined with ‘H-NMR studies an the amide protons, showed that a common feature of all the peptides was a p-turn, and this was confirtned >v t‘he it~afiai>y proline residue. An additional

??Ym-COn%gWdNDn

H-bond

of ihe

was also iden-

tified in Ihe he~a~~\i&75\ (5%. 11). The next Stage was to investigate the corresponding poly-n-pep-

H’ FIG.

;

11. Proposed structure of the HCO.~~-~~a-n\u-ua-~~~~~.

hexapeptide

OLIVER W. HOWARTH

30

and

species are obtained by extraction in acid, followed by fractionation in organic solvents,(229’ and early ‘H and circular dichroism(230’ studies indicated that these molecules showed a low propensity for secondary structure at their N-tennini, where a clustering of basic residues was apparent from sequence studies.‘231) 13C-NMR has proved quite useful for the

investigation of the random-coil regions. Acid extracted histones give 13C-NMR spectra quite atypiFor example, Fig. cal of globular proteins. (1*2*204~232) 12 shows the spectrum of histone H2A (MW 14,500). Typical histone linewidths are much smaller than those of globular proteins, and even the C, and C, regionA also show some correspondingly narrow resonances, implying that a portion of the backbone is mobile. The “structured” regions of the histones

DAVID M. J. LILLEY

have a strong tendency toward self-association, further immobilising the residues in these regions. It has been possible”s’~‘04~232’ to use computer simulation techniques to identify these structured regions. Thus the “constrained” residues are simulated using broadened linewidths and no NOE. An example of the best fit to the H2A spectrum is shown in Fig. 12. The inherent dangers of simulation methods have been very recently pointed out,‘233) although in the following study of histone H2B the authors use essentially the same technique with very similar conclusions. The studies on single histone fractions have been extended to complexes of two and four histones prepared using mild extraction methods.(‘*3~228’ The spectrum of histone “core protein”, a complex containing one molecule of each of the four histones,

(a) h

(b)

100 ppm FIG. 12. (a) Native histone, 10 &/ml, (b) simulation assuming residues l-26 random-coil, (c) simulation assuming all residues random-coil. The identification of the random-coil section was also made by other means. 13C spectrum of native histone H2A. at 45.3 MHz. compared with simulation assuming partial structuring (reprinted with permission from Biochemistry. ““) Copyright by the American Chemical Society).

Carbon-13-NMR of Peptides and Proteins

60

40

20

0

B/wm

FIG. 13. ‘“C spectrum at 22.6 MHz of aliphatic region of core protein (histone tetramer, MW _ 56,000 daltons, 26”C, 100mg ml-‘, neutral pH). The presence of mobile “tails” in the core protein is apparent from the many rather narrow resonances, notably in the u-carbon region.

which is closely related to the core of the chromatin “bead” unit, is shown in Fig. 13. This spectrum, like those of the acid extracted fractions, is strikingly different from those of globular proteins recorded under the same conditions. ‘H-NMR spectra of core protein complexes(228) have shown ring-current shifts, arising from the globular portion of these complexes which must, therefore, have a defined tertiary structure like other proteins. Indeed, from the ‘H-NMR spectra alone, it is far from obvious that the complex is not a normal globular protein. The usefulness of 13C-NMR to this problem, viz. the identification of random-coil regions in the presence of globular domains, lies in the ready ability to study the C, region. Thus, analysis of both the linewidths(232) and the relative Ti relaxation timeso83*232) of CIcarbons has confirmed the presence of random-coil backbone. Further confirmation that these regions are N-terminal comes from examining the changes induced in the 13C-NMR spectra’228’ on specific tryptic removal(234s235)of the N-termini. The results thus derived from 13C-NMR are in agreement with those from neutron scattering(228) and the model for the chromatin bead so derived’236) has important implications for the mechanism of transcription and replication of chromosomes. 7.3.3. 13C enrichmenr. The low natural abundance Of 13C (l.ly,;) may have long term advantages outweighing the disadvantage of adverse signal-tomay be noise ratio, since isotopic enrichment employed. Non-specific enrichment allows the use of lower protein concentrations andjor shorter accumulation times. Enrichment of specific sites in a protein, combined with difference spectroscopy techniques, enables the observation of resonances from enriched

31

atoms only. This has obvious applications in assignment (which have been discussed earlier in this review), and the detailed study of local environmental effects. In this respect “C may be regarded as an ideal “probe” since the substitution is isotopic and hence essentially non-perturbing. Whilst a small kinetic isotope effect might be observed in very special circumstances (though such effects would be predicted to be much smaller than for deuterium substitution), studies of high 13C enrichment in C. utilis 6_phosphogluconate dehydrogenase(237) indicated that minimal kinetic effects were produced. The main obstacle to general use of enrichment lies in the current price of 13C labelled compounds. i3C enrichment is an expensive process involving the cryogenic distillation of carbon monoxide on a very long column. Specifitally-labelled compounds such as amino acids then have to be synthesised from “CO, and the range commercially available is both expensive and incomplete. It is to be hoped that with increased application in biological NMR, this situation will improve. Nevertheless careful experimental design and choice of system are required for efficient employment of label. This inevitably means, for example, the use of micro-organisms in biosynthetic studies. Whilst results arising from enrichment studies have been included in the main text where appropriate, general strategies and. protocol will be considered in greater detail here. 1.3.4. 13C adducts. One approach toward 13C labelling of proteins is in chemical modification (but, of course, here the “non-perturbing” aspect of ‘“C incorporation may well be compromised). The relative simplicity of the technique is attractive but the information which may be extracted is proportionally less. In principle, one can select from a large number of reagents for the modification of various protein side gro~ps,(~~~) although selection will be limited by the ability to synthesise an analogue in which the 13C label remains on the protein. Carboxymethylation by [l or 2 ‘3CJ-bromoacetate has been used in a number of studieso87*239-244) and some selectivity in labelling has proved possible. Thus pancreatic ribonuclease A was enriched at his-119 with [2-13C]-bromoacetate.‘241) In native cyanoonly surface lysine and histidine side-chains are accessible to modification, whereas all such residues, together with methionine groups, become available on unfolding in 8 M urea. By this means, “internal” and “external” labels were introduced. In another specific carboxymethylation, human carbonic anhydrase was exclusively labelled at his-200.(244’ This group was near enough to the enzyme active site to act as a “reporter” molecule, but, importantly, did not result in deactivation. [13C]-methyl iodide has been used in order to methylate protein thiol groups. (2o5) Pig heart glutamate aspartate transaminase has been specifically modified at cys-390 using 13CN(24s) but the resonance thus observed underwent no observable changes upon submyog]obins(l87.242.243)

32

OLIVER W. HOWARTH and DAVID M. J. LILLEY

strate binding. Clearly the general method does not guarantee interesting results. Of course, one possibility for the introduction of a 13C labelled “reporter group” specifically into the vicinity of the active site would be the ‘use of affinity labelling techniques. (246)Thus a competitive inhibitor of an enzyme under study would be “designed” to carry a reactive group incorporating a 13C label. Such an approach is, as yet, untried. A second, if less elegant, way of achieving a similar end would be to use a reagent of general modification pattern with and without substrate present. By this means it would prove possible to prepare enzymes where specific active site residues have been “protected” from modification. The use of difference spectroscopy with the totally-modified enzyme would then reveal the resonance(s) of the active site label. 7.3.5. Semi-synthetic 13C incorporation. Semi-synthetic methods are really an extension of those above, where a given protein has been partially “dissected” followed by a “re-synthesis” using a 13C labelled amino acid. Thus porcine insulin was prepared(247’ in which the usual B chain N-terminal phenylalanine was replaced by either [l-13C] or [2-‘3C]-glycine. Chaiken and co-workers(206--208~248’ have taken advantage of the observation by Richards et al.(24g’ that ribonuclease A may be enzymatically cleaved in a highly specific manner viz: wbtilisin

RNase A residues l-124

RNase S 21-124 enzymatically active + S peptide complex l-20 1 mix

X-ray diffraction studies (24g*250)have indicated that ribonuclease A and its reconstructed analogue have very similar conformations in the solid state. Thus they were able to replace selected amino acids in the S-peptide with “C enriched analogues, followed by reconstitution of the active non-covalent complex. So far phe-8’206) and his-12(208) have been enriched, atid ala-6 replaced by [1-13C] and [2-13C]-glycine.(207’ Solid phase peptide synthesis techniques make experiments of this nature relatively straight-forward. 7.3.6. General enrichment. Non-specific enrichment of all carbons in a protein may be achieved with comparative ease. This is most easily accomplished using a photosynthetic autotroph (e.g. algae) grown on 13C02. Hydrolysates of such proteins may also be incorporated in nutrient media for heterotrophic organisms of greater interest. The advantage in signalto-noise ratio obtained is, however, usually more than offset by extra spectral complexity arising from ‘3C-‘3C couplings. At natural abundance 13C atoms are “diluted” by “C such that the probability of having adjacent 13C atoms is about 10p4. For this reason proton-decoupled 13C spectra are composed essentially of singlets. As the molar ratio of 13C is raised

so is the chance of 13C-‘3C coupling, and spectra rapidly become almost impossibly complicated. Thus general enrichment is dnly useful up to comparatively low levels, e.g. 109; 13C will result in a signal-to-noise ratio gain of ten-fold (or l/lOOth the machine time requirement) whilst Jc- satellites are still only 0.05 the intensity of a singlet single carbon resonance. Such considerations have been treated in some depth in Tran-Dinh et a1.‘125’One possible method for circumvention of the coupling problem is to use twocarbon ‘precursors, where the nutrient contains 50”, highly labelled in one carbon and 503; in the other. In fact this approach has been used in a more specific manner for the labelling of glycine in ribonuclease S-peptide.(207’ 7.3.7. Specijc enrichment. This is probably the most difficult technique but is certainly the method of choice for obtaining useful and biologically meaningful information. Experimental design is of paramount importance. The aim is to label as few sites as possible, and for these to be of functional significance. The economics of 13C enrichment necessitates manipulation of conditions to ensure maximal labelling of the protein of interest, with as little as possible of dilution and scrambling within the metabolic pool. In order to keep resources within the realms of research grants, the use of micro-organisms for biosynthesis is mandatory. Bacteria are the system par excellence. Growth conditions may be manipulated between starvation, stationary and exponential growth phases, and metabolic control is well understood. Thus conditions may be arranged to ensure the most efficient utilisation of 13C. Bacterial genetics are also advanced and the selection of specific mutants relatively easy. The reasons advanced by Hunkapiller et al.(ls4’ for the choice of a-lytic protease as an enrichment system for the serine protease catalytic triad are instructive: (i) The enzyme has a bacterial origin. (ii) It possesses but a single histidine residue. Thus by enriching with [‘3C-2]-histidine these authors were able specifically to label the active site only. (iii) It exhibits remarkable stability toward denaturation and autolysis (the latter being a problem peculiar to the study of proteolytic enzymes). (iv) It is a homologue of mammalian serine proteases (i.e. the enzyme is structurally and mechanistically closely related to important enzymes such as a-chymotrypsin and trypsin). The study of eukaryotic proteins for which no prokaryotic analogue exists precludes the use of bacteria. Yeasts or protozoa may then be used. Tissue culture techniques are a further possibility, although costs are likely to be prohibitive in the reasonably near future. Probably the best long term hope for eukaryotic protein labelling will be in the use of recombinant DNA technology. Thus if DNA coding for a protein of interest is introduced into a bacterial plasmid such that it is transcribed and translated with fidelity, then it may be possible, for example. to biosynthesise

Carbon-13-NMR

specifically r3C labelled antibodies or histones in E. coli.

Probably because of the difficulties involved, few specific enrichment studies of proteins (as opposed to peptides) have been attempted so far. In most cases histidine residues have been enriched (which is almost a pity, considering the relative ease of their study by ‘H-NMR), reflecting their importance in many catalytic mechanisms. Thus histidines have been enriched in cc-lytic protease,“s4) the a sub-unit of tryptophan synthetase of E. COI~.(~~‘,~~~) and in E. coli alkaline has also been phosphatase.(252) [’ %H,]-methionine enriched in a fungal cytochrome-cogs) Such studies should provide the most useful approach towards the ‘%-NMR of proteins, and are likely to become more common in the near future. 1.4. Ligand-binding Studies Many proteins bind small ligands in a highly specific manner. As well as enzyme-substrate (or competitive inhibitor) complexes, high specificity may be observed in other interactions such as receptor protein-hormone, antibody-hapten, enzyme-drug complexes and complexes of “carrier molecules” with their respective ligands. The study of ligand ‘% resonances has considerable merit. Firstly, if the property studied (e.g. shift, relaxation time) pbeys the condition for fast exchange, then provided binding constants are aulailable lower protein concentrations may be offset by using more ligand. Secondly, and of greater importance, r3C enriched ligands may (in general) be prepared with relative ease, and hence the perfect “reporter group” is available, which binds specifically -to the s&t of m@6t p&a&e interest. As sh they are generally non-perturbing although any induced allosteric effects are likely to be of considerable intrinsic interest. In the case of metallo-enzymes, geometric information may be available by study of paramagnetic effects on ligand spectra. 7.4.1. Enzymes. Enzyme catalysed reactions in general proceed through a series of distinct kinetic steps which may be summarised simplistically: E

+

33

of Peptides and Proteins

tion. Transition states are, of course, not amenable to direct study, although an elegant approach is that of “transition-state analogues”, where the ligand is designed to imitate the likely stereochemistry of the transition state and hence hopefully to bind in a similar manner. Rogers and Roberts”37) have studied bound substrate conformation in &chymotrypsin by using ’ 'C)-r.-tryptophan, which is a N-acetyl[carbonyl poor substrate (i.e. effectively a competitive inhibitor). Their results are discussed above in the section on coupling constants in peptides. In general it will be true that experimental error and degeneracy of angle selection does not allow great confidence to be placed in calculated conformations. Observed coupling constant changes probably reflect the selection of a single conformation for binding from the solution population, rather than the forcing of an “unusual” conformation on the bound substrate. The possibility of substrate strain (albeit unlikely) in the ES complex of chymotrypsin has also been investigated using “C-NMR. Robillard et al.‘253) increased the steady state concentration of the complex with L-acetyl [carboxy1 13C]-tyrosine semicarbazide by adding excess reaction product, i.e. semicarbazide. Despite fast exchange conditions, no resonance shift was observed (i.e. less than +O.OSppm) over a bound to free ratio of 0.17-0.70. The authors, on the basis of shifts observed in cyclic ketones, suggest that the absence of shifts indicates a lack of strain in the enzyme-substrate complex. Whilst there is a strong likelihood that this is correct, great care should be exercised in the interpretation of a negative result. Dihydrofclate redz&e-e is an enzyme catalysing the reduction of the coenzyme dihydrofolate to tetrahydrofolate, which is of immense metabolic importance as a single carbon carrier: FolH2 + NADPH -t H+ +FolH,

+ NADP+

Way et al.‘254) synthesised [carboxamido-‘3C]NADP+ and studied its interaction with the enzyme

S=ES=ES====EP=E

enzyme

If natural substrates are used, kinetic intermediates are likely to be too short-lived for observation by NMR, and hence alternative approaches are required. The most common is the study of the binding between the enzyme and a competitive inhibitor of the substrate, which may be assumed to bind in a manner very similar to the natural substrate. Substrate binding may, however, be studied in some cases of multi-substrate reactions, where one or more may be omitted from the solution. These techniques are applicable, in principle, to both “sides” of the reac-

,.P.\.“.R.S.

I2 I---c

from Lactobacillus casei, with and without the other substrate or substrate analogues. In contrast to the above studies, slow exchange Fonditions were observed for enzyme-substrate complex formation, enabling lifetime lower limits to be calculated. The resonance of bound [13C]-NADP+ was observed 1.6 ppm upfield from the free form, but on forming ternary complexes shifts were observed up to 4.33 ppm. Base unstacking and hydrogen-bonding were excluded as major contributors to these effects, and the authors suggest that the most likely explana-

34

OLIVER

W. HOWARTHand DAVID M. J. LILLEV

[4’ and 5’ I3Cl-pyridoxal 5’ phosphate and D-serine dehydratase.@“) Carbonic anhydrase, a zinc metalloenzyme which catalyses the interconversion of CO2 and HCO;, has been the subject of several 13C-NMR investigations of substrate binding.‘258-260’ Feeney and coworkers(25g) bound 13CN- to the human and bovine enzymes and observed resonances for the bound forms which had pH independent shifts of around 2.5ppm from the free form. Comparison with inorganic zinc complexes suggested that the cyanide yas bound to the zinc atom in the active site. Bicarbonate binding to the cobalt bovine enzyme has been studied.(260)Large paramagnetic effects were observed on T, and T, relaxation times, which were removed on FIG. 14. The ‘“C-NMR spectra af 25.2 MHz of (a) a 1 mM addition of inhibitor. The relaxation results were solution of [13CO]NADP+; (b) [‘3CO]NADP+ in the interpreted in terms of two binding sites for the subpresence of dihydrofolate reductase (molar ratio 1: 1); (c) strate, which are inner and outer sphere complexes [‘%O]NADP+ in the presence of dihydrofolate reductase of the metal ion. (moiar ratio 3: 1). The broad resonance between - 2 and Distance calculations have been attempted for a -12ppm in (b) and (c) arises from the protein carbonyl carbons. Sample temperature, 10”. An example of 13C number of enzyme active sites containing a 13C subenriched Iigand binding under slow exchange conditions strate(167*261-264)which have been possible for Mg(“’ (reprinted with permission from Eiochemistry.‘254’ Copyand Zn(“) enzymes. These can usually be isomorright by the American Chemical Society). phously replaced with the paramagnetic Mn’“’ and Co(“) respectively, hence results from 13C, ‘H-NMR, tion lies in polarisation of the carbonyl by a nearby water relaxation enhancement and ESR may be correcharged residue. In a complementary study, Pastore lated. Geometries have been obtained by measureer ~1.“~~) studied the interaction of [benzoyl carbonyl ment of substrate 13C relaxation times, and the appli“Cl-folic acid with the same enzyme. Slow-exchange of relaxation and exchange process conditions were again observed, with a bound shift cation theory.(265-268) This has been well reviewed elseof +2.4 ppm which may result from ring stacking. where.‘6.26g’Thus substrate active site configurations In both studies(4g5) it was observed that exchange carrates become slower on formation of ternary com- have been obtained for ribulose l&liphosphate boxylase, (16’) fructose 1,6-diphosphatase,(261) pyruplexes. vate carboxylase,(262) pyruvate kinase,‘262*263’ enoV H02C lase(263) and pyruvate transcarboxylase.(264) In the Y”Z 7"~ majority of these studies it is calculated that the ‘+ H3P04 T=O + YC-C02H 02” = “,NOAN ), ligands are in the second co-ordination sphere of the H OPO,” NH, metal ion, giving distances of OS-O.8 nm. In view of ureidoruecinic the difficulties inherent in estimating several of the carbamyl arpartic acid acid phosphate parameters involved, for example in the selection and Aspartate transcarbamylase catalyses the first step estimation of correlation times, greater precision than this is probably not possible. Approximate orienin pyrimidine biosynthesis, and is an important metatation of the substrate has been calculated for some bolic control point. This is accomplished via allosteric effects resulting from a regulatory subunit on effector enzyme substrate complexes by comparing relative relaxation rates of different carbon atoms. [l-13C] binding. Roberts et a1.‘256) have studied binary and ternary enzyme substrate complexes using [13C] car- and [2-13C]-pyruvate have been bound to pyruvate bamyl phosphate and the catalytic subunit from E. kinase 9(262,263)pyruvate carboxylase(262) and transcarboxylase. (264)In pyruvate-Mn(“‘-pyruvate carboxycoli. Whilst only a small shift was observed for the binary complex, addition of succinate produced a lase(262) the T, of the pyruvate carbonyl was reduced -0.63 ppm shift at pH 7.0, interpreted in terms of a by a factor of 3 compared to the carboxyl carbon when the values were normalised with respect to protonation in the active site. Other dicarboxylic acids produced rather smaller effects. The conclusions pyruvate-Mn”‘). However, on forming the quaterare strongly supported by the results of binding a nary complexes pyruvate-Mn”“-phosphate-pyruvate transition state analogue, [amidocarbamyl 13C]-Nkinase,(262) this distinction was not observed, indicat(phosphonacetyl)L-aspartate (PALA), to the enzyme. ing a different substrate orientation in the active site. This resonance is strongly sensitive to ionisation of In transcarboxylase,(264’ pyruvate was shown to form second sphere complexes with both Co”‘) and C@‘) the phosphonate group, and again a downfield shift of 0.8 Hz was observed for the bound species. (which have different binding sites). Furthermore the Further indications of shifts in bound ligands orientation of pyruvate was similar to that in pyrecome from study of Schiff base formation between vate kinase.

35

Carbon-13-NMR of Peptides and Proteins Zens et ~1.‘~~‘) have used 13C Ti measurements supplemented by 2H T2 values to investigate the binding of nicotinamide adenine dinucleotide and adenine 5’ monophosphate to chicken-breast M4 lactate dehydrogenase. The binding of NAD appears to involve the adenine ring in preference to the nicotinamide moiety. 7.4.2. Lectins and carrier molecules. Substrate binding to non-enzymic proteins is less complicated since no chemical reaction with the ligand results. Plant lectins bind to cell surface carbohydrates causing agglutination of oncogenically-transformed cell~.(~~‘) They are thus highly useful probes of cell surface effects, and also the specificity of ligand binding’272’ has resulted in comparisons with antibodyantigen reactions. Several groups have studied the binding of “C enriched saccharides to the lectin concanavalin A,. and, as with some of the enzyme studies above, used metal ion replacements to obtain distances in the binding site. Sternlicht and co-workers have studied the binding of uniformly 14% 13C labelled a and /I methyl D-glucopyranosides to concanavalin A’273-275)with zinc, cobalt and manganese present. Measured T, values were used to calculate bound geometries. Linewidth temperature dependences indicated different exchange rates for the two anomers, and calculated metal-13C distances were consistent with different binding orientations. Villafranca and co-workers(261*276) have performed natural abundance “C studies on the binding of a-methyl glucopyranoside to concanavalin A. They have also used T1 measurements with the Mn”‘) lectin, but have thereby calculated a different conformation from that of Sternlicht and colleagues. The average metal-carbon distances are 1.Onm and hence calculated conformations are unlikely to be accurately determined since most carbon-metal distances will be with 0.1 nm of this, especially considering the “damping” effect of the sixth root. Correlation times have been estimated from the relaxation times of the diamagnetic zinc protein complex and this may introduce a further error. Indeed, Fuhr et al.“65’ in a ‘H-NMR study of a methyl n-mannopyranoside to concanavalin A have used measurements at two frequencies to estimate correlation times with less ambiguity, and have calculated metal-ligand distances of 2.15 k 0.12 nm. Transferrin is important for iron transport across the reticulocyte membrane. Harris et al.(277’ have studied the binding of carbonate ions to its two iron atoms. They have been able to show that the sites are inequivalent, but distance calculations have not been successful. Another transport protein studied by 13CNMR has been serum albumin. The binding of detergent alkyl sulphates has been examined(278’ and an attempt made to relate bound shifts to the hydrophobicity of the environment. Relaxation times indicate that the mobility of the terminal methyl groups of the alkyl chains are little affected by binding to the protein, and thus the bound environment is rather micelle-like.

Perhaps the most important group of carrier proteins are the haem proteins, notably myoglobin, haemoglobin and the cytochromes. Interactions with “C ligands, particularly i3C0, have been extensively studiedo93*279-2g5’ and these have been well reviewed else&here.‘@ Ligand binding studies in haemoglobin using r3C0 have enabled the study of the different binding sites, the interactions between them, Le. homotropic allosteric effects, and comparisons of active site environments between structurally and genetically distinct haemoglobins. 1.4.3. Antibody-hapten interactions. Goetze and Richards(2g6) have recently shown that when phosphorylcholine binding mouse myeloma protein M603 binds its 13C enriched hapten, p hosphoryl (Me-13C) choline, there is rather limited hapten immobilisation although the hapten is in fairly slow exchange. 1.4.4. A warning. Although ligand-binding studies are of immediate value, particularly to enzymologists, they need to be approached with some care. Blumenstein and Hruby,‘32’ in a study of the binding of i3C-labelled oxytocin and arginine vasopressin to neurophysins, found that the substrate T2 was very strongly dependent upon the precise physical conditions (especially the temperature) under which the spectrum was obtained. REFERENCES 1. R. A. DWEK, N.M.R. in Biochemistry: Applications to Enzyme Systems. Clarendon Press, Oxford (1973). 2. K. WHICH, Nuclear Mqmetic Resonance in Biological Research-Peptides and Proteins. North-Holland, Amsterdam (1976). 3. H. W. E. RATIXE, Prog. Biophys. Mol. Biol. 28, 1 (1973). 4. E. M. BRADBIJRY, P. D. CARY, C. CRANE-R• BIN~~N and P. G. HARTMAN,Rev. Pure Appl. Chem. 36, 53 (1973). 5. P. F. KNOWLES. D. MARSH and H. W. E. RATTLE, Magnetic Resonhnce of Biomolecules: an Introduction to the Theory and Practice of N.M.R. and E.S.R. in Bioloaical Svstems. Wilev-Interscience, New York (1976j. _ 6. G. C. LEVY (ed.) Topics in C&on-23 N.M.R. Spectroscopy, Vol. 2, PP. 1 and 179. Wiley-Interscience (1976). 7. D. W. URRY and L. MITCHELL,Biochem. Biophys. Res. Commun. 68, 1153 (1976). 8. M. KAINOSHOand H. KONISHI,Tet. Left. 4757 (1976). 9. P. C. LAUTkXBUR, Appl. Spectroscopy 24, 450 (1970). 10. D. G. GILLIESand D. SHAW. Ann. Reo. N.M.R. Spectroscopy 5A, 560 (1972). . and E. OLDFIELD,Bio11. A. ALLERHAND,R. F. CHILDJXRS chemistry 12, 1335 (1973). and P. DULLENKOPF,Rev. Sci. Instru12. H. J. SCHNEIDF,R ments 48, 68 (1977). 13. D. I. HOULT and R. E. RICHARDS,J. Msg. Resonance 24, 71 (1976). 14. A. C. MCLAUGHLM, P. R. CULLIS, M. A. HEMMINGA, D. I. HOULT, G. K. RADDA,G. A. RITCHIE, P. J. SEELEY and R. E. RICHARDS,FEBS Len. 57, 213 (1975). 15. W. NIEDERBERGERand J. SEELIG.J. Amer. Chem. Sot. 98, 3704 (1976). 16. R. DESLAIJRIERS,R. A. KOMOROWSKI,G. C. LEW, A. C. M. PAIVA and I. C. P. SMITH, FEBS Left. 62, 58 (1976).

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Carbon-13-NMR

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