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Amide proton exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution
,I. Mol. Rid. (1982)
Amide
160, 343-361
Proton Exchange and Surface Conformation Basic Pancreatic Trypsin Inhibitor in Solution
Studies with Two-dimensional
(Received
Nuclear
11 February
Magnetic
of the
Resonance
1982)
A norc~l approach for studies of amide proton exchange in proteins is presented. It relies on measurements of the amide prot,owC” proton cross-peak intensities in the two-dimensional homonuclear correlated ‘H nuclear magnetic resonance spertra. The protein is dissolved in ‘Hz0 and the solution is exposed to the conditions of p2H and t,empwature where the exchange rates are to he measured. After variable int,ervals. the amide proton exchange in a sample of this protein solution is quenched by lowering the temperature and possihl,v hg p2H variation, and a C’OSYt spectrmn of this sample is then recorded. Comparison of the NH-C”H cross-peak int,rnsities in t,he specka recorded after different exchange t,imes yields exchange rates for the individual amide protons. The main advantage compared to previously described techniques is that a much more complete set of individual arnidr proton exchange rates can he ohtjained. In the hasir pancreatic trypsin inhihitor. where all the amide proton rcsonanccs were previously individualI> assigned. quantitative exchange rates were obtained for 38 of the total of 53 hac*kl)one amide protons. and for I? addit,ional protons lower limits for the exchaltge rates lverr estahlished from comparison of the COSY spectra recorded in H,O and in 2H20. Proton exchange data lvere thus for the tirst time obtained for numerous peptide groups t,hat are located near the prot,ein surface in the sin&~ crystal struc%ure of RI’TT. For sonle locations on the protein surface. it appears that the amide prot,on exchange rates cannot he cwrrrlated readily wit,h the static, awrssihle surface areas in the cr,vstal structure.
1. Introduction stability of biologically active spatial protein structures relies on a complex interplay of a multitude of weak, non-bonding interactions among different atoms of the polypeptide chains and between the polppeptide chain and the surrounding medium. The lat)ter may, for example, be an aqueous solvent, an ordered lipid matrix The
344
(:. b'A(:NEK
ASI)
K. Wi;'FHRICiH
in biological membranes, or the ordered aggregation in single crystals used for Xray studies. Since the contribution of each individual non-bonding interaction to the free energy that stabilizes the protein conformation is typically of the same order of magnitude as the thermal energy at temperatures near 300 K, these “secondary bonds” are constantly broken and reformed. As a, result, protein molecules are highly dynamic structures. On the one hand, since they depend on intramolecular interactSions and on interact,ions wit,h their environment. protein conformations adapt readily to local changes of the polypeptide covalent structure and to changes of their surroundings. Examples are the conformational transition from trypsinogen to trypsin (Huber. 1979). t,he local conformation change in carboxypeptidase A upon substrate binding (Quiocho & Lipscomb. 1971). the quaternary structure transitions in tetramerir hemoglobin (Perutz, 1970) and quite generally the potential adaptability built into multi-domain protein structures with flexible hinge regions (Huber. 1979: Schulz Br Schirmer, 1979: Richardson. 1981). On the other hand. protein mole:cules in thermodynamic equilibrium situations undergo time fluctuations about, an average set, of atom co-ordinates. These st,ruct,ure fluctuations cover a wide range of frequencies. amplitjudes and energies of activation. They have been observed, for example. in nuclear spin relaxation measurements (Allerhand rt al., 1971 ; Ribeiro et nl., 1980; Richarz et al., 1980), aromatic ring flips (Wagner et ul., 1976) and amide proton exchange studies (Hvidt & Nielsen, 1966: Englander et al., 1972: Richarz et al., 1979), and were extensively investigated by molecular dynamics calculations (Karplus Qi McCammon, 1980). This paper describes amide proton exchange measurements wit,h the use of two-dimensional nuclear magneticresonance. The results of these experiments bear on both the adaptation of the molecular conformation to changes of the protein environment and the time fluctuations of the protein about an equilibrium set of atomic co-ordinates. In the above-mentioned, well-documented examples of conformational changes upon variation of the protein covalent’ structure or the protein environment, the initial and final states of the protein could both be studied by X-ray methods in single crystals (Perutz, 1970: Quiocho & Lipscomb. 1971 : Huber, 1979). Different experimental techniques must, be applied t,o investigate how the molecular conformation adapts to the change of environment, when a protein is transferred from single crystals to a non-crystalline state. Such a technique should be capable of determining the polypeptide conformation with comparable detail to t.hat of the single crystal X-ray structures. We have recently proposed that this cbould be achieved with ‘H r1.m.r.t experiment,s (Wiithrich of ai.. 1982), and as a first, fundamental step complete individual assignments werp obtained for protein ’H r1.m.r. spectra (Rilleter rt al.. 1982: Wagner & Wiithrich. 1982: Wider et al., 1982: -4rseniev et al., 1982). In this paper. 2D n.m.r. is used to investigate the exchange with the solvent of all the backbone amide protons in the basic pancreatic trypsin inhibitor. Combined with the previously obtained resonance assignments. these data serve t,o probe the molecular surface of KPTI in solution. which will then be correlat,ed with data on the crystal structure. t See footrlotr to p 343
sITRFACE
STRI-CTlTRE
OF
BPTI
IN
:Lki
SOLl”~lON
Amide proton exchange measurements have long been applied for studies of internal fluctuations in proteins (for reviews, see e.g. Hvidt & Nielsen, 1966: Englander et al., 1972), and already in 1958 Saunders & Wishnia demonstrated that proton exchange between proteins and solvent 2H20 can be observed by n.m.r. While other techniques for kinetic studies of specific groups of protons have been described (e.g. Rosa & Richards, 1979), the potentialities of n.m.r. for measurements of individual amide proton exchange rates appear to be quite unique (Wtithrich. 1976). With the experiments described in this paper, one obtains for thca first time a complete data set, i.e. the individual exchange behaviour ten be charact,erized for all the backbone amide protons in a protein. This should be of great value with regard to further clarification of the mechanistic aspects of amide proton exchange from globular proteins (see e.g. Wagner & Wiithrich, 1979: Hilton et al., 1981), and one can hope to obtain a more complete view of the internal “breathing modes” (Hvidt & Nielsen. 1966) than has hitherto been possible.
2. Materials and Methods Basic pancreatic
trypsin
inhibitor
(Trasylol Cm , Havrr Levrrkusrn)
was obtained
from the
Farbenfabriken Bayer AU. The solutions used for the n.m.r. recordings contained 0.02 MHI’TT and the pH was adjusted by the addition of minute amounts of HCl and NaOH. whereby in the 2H20 solution the pH meter readings were used without correction for isotope effects (Kalinichenko, 1976; Bundi & Wiithrich, 1979). For the experiment in Fig. I. a mixed solvent of 90% Hz0 and 10qb ‘HzO. pH 4%. was used. so that all the backbones amide proton resonances were present in the spectrum. To observe a maximum number of it is important that from t,he first contact amide protons in a protein solution in ‘H20. between protein and ‘H,O the p*H IS near the p2H minimum for proton exchange (Wiithrich &r Wagner. 1979). Therefore, to prepare ‘H,O solutions of BPTl for the proton exchangra measurements. the protein was first dissolved in H,O and the pH adjusted to 3.5. h’ext. RPTT was Iyophilized and dissolved at 24°C in 2H20. The resulting p2H of the RPTT solution wa,s in all experiments in the range 3.5 k 0.2. \\,hich was then adjusted to 35 with the use of a combination glass electrode. The measurements in this paper were all done with homonuclear 2-dimensional (21)) correlated spectroscopy (COST) (Aue et al.. 1976). COSTi uses t,he pulse sequence (Aue cjt rrl.. 1976; Nagayama et frl., 1980: Wagner P/ nl.. 1981): 190’ -t,
-90”-t,],.
(1)
where t, and t, are the evolution period and the observation period, respectively. To obtain a 21) n.m.r. spectrum, the measurement is repeated for a set of equidistant t, values. To improve the signal-to-noise ratio and to eliminate experimental artefacts. )L groups of 16 recordings with different phases were added for each value of 1, (Nagagama ~1 nl.. ISi!!. 1980). At the end of each recording. th(s system was allowed to reach equilibrium during a fixed delay of 1.2 s. The spect,ra were recorded at 500 MHz on a Bruker WM 500 spectrometer. The spectrum in Fig. I was obtained from 512 measurements. with t, values from 0 to 47 ms and 2048 points in t2. To reduce the observation time, the spectra in 2H20 were obt,ained from 2% measurements, with t, values from 0 to 24 ms and 1024 points in t,. To end up with a 1024 x 1024 point data matrix in the fmqucnc? domain. which corresponds to a digital resolution of 5.7 Hz/point, the time domain matrix was in all experiments expanded t,o 2048 points in t, and 4096 points in t, by “zero-filling”. Quadrature detection was used for detection of the individual free induction decays, with the carrier frequency at the low-field rnd of the spectrum. For the measurement in H,O. the
346
C:. LZ’A(:NER
ANI)
Ii. b\‘i”PHKIC:H
solvent resonance was suppressed by select.ive. continuous irradiation at, all times rxwpt during data acquisition (t2 : Anil Kumar rt nl., 1980). Prior to Fourier transformation. thr time domain data matrix was multiplied in the t, dirt&on with a phase-shifted sine bell. sin (n(t+t,)/t,), and in the t, direction wit,h a phasr-shifted sine-squared bell. sin’ (~(t c/,)/t,). The length of the window func~tions. t,. was adjusted for the hells to reach zero at the last experimental data point in the t, or t, direction. respwtively. The phase shifts. t,if,, were l/32 and l/64 in the t, and t, direction. respectively. The Hz0 spectrum in Fig. 1 \vas further improved by symmetrization (Raumann PI trl.. I!)81 ). The absolute value presentation was used in all the experiments. Quantitative measurements of the amide proton clxchxnge rat’es were made at 36’ C and 68°C’. The *H,O solutions of BI’TT used for t,hrsc> t~xp(~riments were prepared at 24’C. Immediately after the protein ha,d been dissolved alrd the p2H adjusted to 3.5. t,he solution was heated to the desired exchange temperat.ure for a certain length of time. Then it MBS cooled to 24°C’. and the C”OXY spectrum was rewrded during 11 h at 24°C. A new sample \~as prepared for each of the measurements with rxrhangtx time-s at 36°C’ of 0. 10. 240. 660 and 1720 min (Fig. 3). rlfter the completion of the n.m.r. rwording. the sample of the 1720 min experiment was further used for all t,ho remaining measurements with longw exchange times (see Fig. 3), where the excahangt, during the 11.mr. rerording at 24 C is Ilegligibly small compared to the exchange during the kvaiting timths at 36°C’. Similarly. IIe\v samples were prepared for the individual measurements at 68-C’. \vhicah wer(’ recorded after uaitillg t#imrs of 0, 5. 10 and 15 min. The same sample as for tht, I,5 min expc~riment, was further used for 2 additional measurements lvith exchange times of 60 and 120 min. To measure resonance intensities. cross-wctions through the COS\’ spectra wre plotted (Nagayama rt al.. 1978) and the height of the individual peaks was measured. For all the measurements. the spectra \vrre recorded with identical instrument settings alld the datta were handled in identical \vays. The peak heights ilr each spectrum were measured relatiw to t,hose of the well-known and well-separated low-field C’H-C”H cwss-peaks of Tyr2l and Cys30 (Wagner & Wiithrich. 1982). Since wit,hw C” nor (‘” protons exc*hange wit.h *HzC). these two peaks have intensity I in each spec%rum independent, of the exchange time and exchange temperat,ure. The rate constants in Tablr 1 were obtairrcatl from a nolr-linear least-squaws tit of the c~xperimental data to an c~xponential function. ;2n error analysis was performed for all those protons. for which the time dependence of the peak illtensity could be measured at 4 or more points. For 19 amino acid residues in KI’TI. the amide proton rxchallgc is so fast that their NHC”H COSY cross-peaks were absent, already frortl the spectrum recorded after the shortest exchange time used at 36°C. To obtain furt,hcr information OII these exchange reactions. new KPTI solutions in ‘H,O were prepawd at 4’(’ arId the p2H n-as again adjusted to 3.5. .L\ COSY spectrum of one of t,hesc samples \vas rw~ordrd iI1 12 h at 1O’C immediateI?- after sample preparat.ion. The seco~ld sa.mple \vas heated to 3BC‘ for 10 min twfort- a (Y)S\. r(wjnance assiglunerits at spectrum was recorded in 12 h at lO,C. For t.hrsr experiments. 10°C were obtained with th(* previously dcs~rilwd t.whniques (Wagner 8: Wiit.hriqh. 194’2).
3. Results In two-dimensional correlated spect,rosc*opy. one obtains a square array of resonance peaks in the w1-w2 plane (Fig. I ), In the complete COSY spectrum, the to the normal, one-dimensional so-called diagonal peaks with w1 = w2 correspond spectrum (see e.g. Nagayama et al., 1980; Wagner & Wiithrich, 1982) (the diagonal peaks are not shown in any of the Figures in this paper). Any two diagonal peaks at w1 = w2 = wA and w1 = w2 = wx that originate from protons linked by scalar spinspin coupling (J-coupling), are connected by a pair of “cross-peaks” located in symmetrical positions with respect t.o the diagonal at (wl = wA, w2 = wx) and
SI’RFACE
STR[lCTURE
OF
BPTI
IN
347
SOl,L-TION
w2 (p.p.d
r-
8 I
9 I
IO I
7 I
COSY Hz0 80 oc
3
6
I 9
I 8
I 7
-6
WE (p.p.m.)
FIN:. I. Symmetrized (Baumann et al., 1981). absolute value 500 MHz ‘H COSY spectrum of a 002 VI solution of RPTT in a mixed solvent of 90(X Hz0 and loo/* *H#. pH 46. at 80°C. The spectrum was recorded in approx. 24 h, the digital resolution is 5.3 Hz/point. The entire spectrum was presented previously (Wagner & Wiithrich, 1982). Here. the region (wl = 1~5t06~0p.p.m..w2 =6~6tolO%p.p.m.). is shown in a stacked plot representation in the lower part of the Figure, which affords a “3 dimensional” view of the spectrum. and as a contour plot in the upper part. The temperature of 80”(’ was chosen to avoid the appearance of cross-peaks with the labile protons of arginine and Iysine side chains. which show up at lower temperature at this pH value. The spectrum thus contains only NH-C”H cross-peaks. whereby all the amino acid residues can be observed except Argl. Gly37 a,nd the 4 prolinc residues (Wagner & Wiithrich. 1982).
348
(:.
\Y.i(:NEH
ASI)
IC
lVi’THKI(‘H
one works with a mac~romole~ule sucsh as a protein. a ( Wl = wx> w2 = wA). When single (‘OSY spect,rum can provide information on all t,he proton-proton fJconne&ivities in t,he molecular structure. The presentj applic*ation of (‘OSY for studies of amide proton rxchangt, rates relies on measurrments of’ the time dependence of the intensities of the pofylwptide harkbone amide I)roto~~A” proton cross-peaks. which we ha,ve previously c+alft~l the “n.m.r. fingerprint of the protein Aructurr” (Wagner & iViithric>h. 1982). In a protein C’OSY spwt’rum rrwrded in H,O at pH near 3.5. each amino acid residue gives rise to one SH- (‘“H cross-peak. Exceptions are the gfycine residues. which give in general two cross-peaks. t,he N~tt~rminal residue. where t,hr amino protons usually exchange too rapidly t)o lw ohserved (Scheinblatt Cy: Rahamin. 1076: Bundi R- Wiit’hrich. 197!1) and. of course. the prolinc residues (LVagncr & Wiithrich, 1982). The TL’H-C”H cross-peaks are usually all cwnt,ained lvithin a limited spect)ral region, which contains no 01’ rt3r.y fbw othrr cmss-peaks, tlt~pending on the conditions of the experiment. L2’hcn the arnino acid sequenw is known. inspection of t’his region of t,h(L (‘OSY spectrum thus pro\-ides a rapid clheck of whether all or most of the amino acid residws c.an lw observed and their resonances resol\-ed in the 21) n.rn.r. spectra. For KPTI _ all N HP(‘“H cwss-peaks are located in the spectral region (wl = 1.8 to 5.8 p,p.m.? w2 = 6.7 to 10.5 p,p.m.). and at the pH and t’emperature of the experiment in Figure l this region contains no ot’her crosspeaks (Wagner & LViithrich. I!X%Z)j-. Figure 1 presents an almost wmplrte The NH-(‘“H cross-pwks are fingerprint of t,he BPTI amino acid sequcnw. resolved for 52 rrsidurs. i.e. all rxcrptj Ar’gl , (:lyX and t,he four profine residues. and all the peaks were previously assigned t,o specific residues in the amino acid sequence (N’agner B Wiithrich. 1982). X reduced fingwprint of the protein strwturc is afforded hy the same region of a (‘OSY spectrum recorded in 2H20 (Fig. 2). SHY (‘“H (aross-peaks are seen only fol those residues for which the amide proton exchanges rela,tively slowly \vit,h 2H of the solvent. Inspection of Figure 2 reveals that instead of the cross-peaks for 52 residues seen in Figure 1, only 33 residues are manifested. The peaks of these 33 residurs are identified in Figure 3. Individual assignments for all the backbone arnide prot,ons of BPTI have been described at 68°C and pH 4.6 (Wagner C% LViithrich, 198%). For all the protons observed in 2H20, the assignments at 24°C’ and p2H 3.5 were independently established with the same techniques (some assignmen& at 21°C’ are documented 1)~ \Vagner it crl., 1981). (‘ompared tjo
up (p.p.m.)
COSY 24 T
7 I
8 I
9 I ‘H,O
;P
6
I 9
I 6
1 7
w2 (P.P.~)
FIG:. %. Ahsolute value 500 MHz ‘H POSY spectrum of a @02 M solution of KI’TI in ‘H,O. pZH 3.5 at 24 (‘. Irnmrdistety twfore the n.m.r. experiment. the protein was dissolved in ‘Hz0 at 24 (‘. Thcs solution vxs thrn kept at 36’C for 10 min and then cooled again to 24’C. at which trmperature the spectrum was recorded in 12 h. The digital resolution is 53 Hz/point The same spectral rqion is show11 as in Fig I, Only the NH-C”H cross-peaks with the stowty exchanging amide protons (Wiithrich k The vertical noise bands lwtwrrtr 6% and \Vagnrr. 1079: Wagner & Wiithrirh. 198%) are observed. i.5 p,p.m are “tails” of the strong aromatic signals in thv adjacent spwtral wgion.
(:.
I+‘A(:NER
AN11
K.
Wi;THRlCH
Y23
“‘O
N24
0 F45
min
1720
mm
660
240
mln
0
T32
0
A27
-
I’
B
IO mm
, IO
9
0 c30
, 8
i
I 7 W* (pwn.)
FIN:. 3. Absolute value 500 MHz ‘H COSY spectra of 0.02 M solutions of BPTI in *HZ0 recorded at different times after the protein was dissolved. The solutions were freshly prepared at 24°C and then kept at 36°C to allow exchange of protein amide protons with *H of the solvent. At the times indicated in the Figure, a particular solution was cooled to 24°C’ and a spectrum was recorded in 12 h. The digital resolution is 5.3 Hz/point. Compared to Fig. 2. the size of the plot was reduced to the region (wl = 3.6 to 60 p.p.m., (02 = 66 to 10.8 p.p.m.), cutting off the cross-peak of Cys51 at (wr = 1.85. w2 = 7.05 p.p.m,) and 1 of the 2 cross-peaks of Gly36 at ( w1 = 3.4, w2 = 8.6 p.p.m.). Furthermore, the vertical noise bands between 66 and 7.5 p.p.m. have been covered with white paint. The peaks that disappeared in the course of the experiment are identified in the last spectrum. where they can be observed readily. Thus this Figure affords a qualitative survey of the exchange rates (for quantitative data. see Fig. 4 and Table 1). The peaks that did not disappear within 80,000min are identified in the last spectrum. The disappearance of the C51 cross-peak, which is outside the spectral region shown. is indicated with an arrow in the spectrum taken after 5880 min.
w2 = 6.23
p.p.m.
116 G26
1
I
I
r
---. 7
J I
6
5
4
3
6
5
4
r
3
I
I
7
-I
3
6
5
4
3
6
w,
5
1
I
4
3
(rw0.m.)
I+:. 4. Vrrtioai c.ross-sert~ions of the spectra shown in Fig. 3 taken at 1 different positions along thr (u2 axis. At thr top of the Figure. those residues are idwtified for which w2 coincides with w2 ofthr crosssection The cross-peaks of the residues indicated in parentheses are located so close to the c-ross-sections that tails of the peaks are observed in thvsr presentations.
Figure I. t,hr cross-peaks in Figures 2 and 3 are considerably broader. This is a consequence of both the lower temperature and the smaller number of t, and l2 values used to record the spectra (see Materials and Methods). For the 33 residues that can be observed in Figure 2, the time-course of the crosspeak intensity was followed over a period of approximately two months (Fig. 3). For the quantitative measurements, cross-sections of the COSY spectra (Nsgayama d (11.. 1978) were used as illustrated in Figure 4. It seems worthwhile t,o point out the excellent signal-to-noise ratio achieved with the present experiments (Fig. 4) and to emphasize that the spectral analysis relied on measurements of the peak heights relative to peaks t,hat are known not, to vary with time (see Materials and Methods). For four residues, only upper limits for the exchange rates at 36°C’ were obtained (Table I), since the peak intensities were essentially unchanged after 83040 minutes (Fig. 3). For 26 residues, t’he time change of the peak intensity could be followed in four or more of the spectra in Figure 3. For these, error limits for k, (36°C) ranging from +3 “/b to _+ lo’?&, depending on the proton, were computed (Table 1). For the remaining three residues, less than four data points were obtained. so that error limits for k, could not be established reliably (Table 1).
352
(:.
WAGNER
ANI) T.4Irl,l~;
Rate constants
Amino acid residue
\V~THKICH
1
10-” ,min-’ , + loo/,) for thr exchange oj’ i~~divid~ual protons in BPTI at p2H $5 and 36°C and &I”(
k,(in
backbonr
amidr
k,(36Yyt
Argl Asp3 Phe4 CyvsT, I,eu6
n.o 10 PHI 0.3 1 023 I2
GlU7
TyrlO Thrl I Gly12 Cysl4 LyslS Ala16 Argl7 Ilel8 lIeI Arg20 T+2l Phe22 T)T23 Am24 Ala25 Lys26 Ala27 Gly28 Leu29 (‘\40 Gin31 Thr32 l’he33 Val34 T)Y% Gly36 my37 f’ys3X Arg3’) Ala40 Lysll Arg4P Am43 Am44 Phe45 Lys4ci Ser47 Ala48 Glu49 Asp50 Q-&51 Met52
Ii.
-100
* 1.1
23: f
,.,;
87
f 00022 < OAO4
-100 -4, tr ’ I3
091 1 - 80 0aoO48 43
om1o 3.1 MO44 (FOTO 11.0. - 80
f 0.5: - 100 om75 04013
f -100 f -100
II.0. - 330 f 93 43 14 93 f
f 032 0,057
f 65 f 7.1 56 5.9 62 12 f - 32:)’ 52 9% f
11 210 ti+?i 140 I0 50 n.o. f t 1’ 180 f f 3i 20 f f f f - 5:; II
37.000 51 .OOO 1 I0.ooo <56,000 39,000 %4.000 39.000 40,000 88.000 69.000 .56.000 35.000 36.000 30,000 35.000 44 .ooo 28.000 28.000 88.000 78,000 35,000 .56 000 8o:ooo 59.000 88.000 78.004~ I 23 .ooo 44.000 36 .ooo 24.000 99 .Ooo 16R.000 li5.000 69.000 .i6,000 35.000 70.000 I 39 .ooo 196.ooo 62.000 37,000
174,000 5N ,000 99.000 i2.000 144.000 56 .ooo
+ + + +
+ + +
B
i
B
+ +
a
+ +
+
+ + +
4
SITRF.4(‘E
STRI’(‘T1’RE
OF
BPTI
IX
SOLI’TIOS
xi3
TAHLE 1 (continued In In solution k,(36”(‘)t 0.07 1 0~40 0~036 2.1 f f
Accessible surface area$
k,(68”r)t 16 33 20 79 f f
3.5 .ooo 174.000 174,000 156 . 000 158.OOO 63.000
04 0.0 04 0.0 3.0 11.7
the
crystal
Hydrogen bonding + + + +
structure
)I
Regular secondary strurture
7
\ 1 \ 1
For ti8 (’ and p*H 3.5 the intrinsic exchange rates, k,,,, are also given. Further. the following properties of the individual amide protons in the crystal structure of BPTT are listed. Accessible surface area, hydrogen Ironding and. where applicable. location in a polgprptide segment which forms regular secondary structure. I- The numtwrs give k, in IV3 min - 1 k IO’&. The sign - in front of a number indicates that less than 4 experimental points were measured and therefore no error analysis was warranted. n.o.. not ohserved. Indicates that the NH resonance of this residue could not be detected in the COSY spectrum. f indicates that the amide proton exchanged too rapidly to he seen in the POSY spectrum recorded at 10°C (see the text). ‘I’hv following limits for the exchange rates of these protons were estimated: k,(36”C) > 0.t min-’ : k,(68’(‘) > 5 min-’ (see the text). : Intrinsic rate constants at p2H 3.5. which reflect inductive effects of neighbowing residues. \vew valwlated according to the rules of Molday rt cl/. (1972) starting from equation (2) of Englander rt crl. (1972). To obtain these data, pK, values of 3.4. 3%. 3.8. 3.0 and 29 were used for 4sp3. Glu7. Gtu49. Asp50 and thv C-terminal Ala58. resprctivel,v (Wiithrich & Wagner. 1979). k,,,,(BS”C) in IV3 min-‘. 5 Acwssihlr surface area as defined by Lee & Richards (1971). The calculations for BPTI are described in (‘hothia & .lanin (tR75). The data used here are taken from this reference and from a complete listing of t,tu, solvcrlt acwssihilities for all the atoms in BPTI, which was kindly given to us hy Dr C. Chothia. /I A + sign indicates that t,he amide proton of this residue was assigned to a hydrogen bond in the rrtinc~d (,rystal structure (Deisrnhofer & Steigemarm. 1975). * 1 and p, resprctivcl~.indicate that the amide proton was found t,o tw in a hydrogen bond that is part of an \-hc>lix or an antiparallel p-sheet in the refined crystal structure (Drisenhofer & Steigemann, 1975).
(‘orresponding experiments were done at 68°C’. The exchange at this temperature could be followed for 31 amide protons (Table 1). For 28 residues, k,(68”C) could be established wit,h error limits of + lOo/,,, and for three residues. approximate exchange rates without error limits were obtained. Addit’ional rxperiments were used to further discriminate among t,he 19 amide protons that, exchange too rapidly to be seen in the first spectrum recorded in the experiment of Figure 3. A 2H,0 solution of BPTI was prepared at, 4°C and immediat,ely thereafter a COSY spectrum was recorded at 10°C in 12 hours. Figure 5 shows that, compared to Figures 2 or 3, five additional residues could thus he observed. In a different experiment, where an identical sample was heated to 36°C’ for ten minutes before the COSY spectrum was recorded at IO”C, two of these ti1.e cross-peaks were too weak to be seen and the other three had lost much of their intensity. From these observations, we estimated that k,(36”C) - 0.1 min-’ for the five prot)ons identified in Figure 5. For the 14 prot,ons not seen in the experiment at IO’C’. we expect accordingly that k,(36”C) > 0.1 min-’ (Table 1). (See Discussion for furt,hrr details on how these values were obtained.)
3.54
(:. WA(:NER
ASI)
K. M'i'THRIC'H
4. Discussion (a) 21) TZ.m.~.
for
studies
of nmidr
proton
rxchanp
rates
The main advantage of the presently described use of C’OSY for studies of amide proton exchange in proteins is the improved spectral resolution, which allows observation of separate peaks for nearly all amide protons in RPTI. Compared to corresponding unidimensional n.m.r. experiments. the overlap of amide proton resonances with those of the aromatic protons is completely removed, and the resolution between different amide protons is great’ly improved, With unidimensional experiments at 360 MHz, quantitative exchange data at’ temperatures near 3ci”C previously obtained for 16 individually assigned arnide protons of BPTI (Wiit,hrich Sr Wagner. 1979: Richarz et 01.. 1979). This already compared favourably with the amount of information on individual exchange rates that can be obtained from experiments other t)han n.m.r. (see e.g. Hvidt Rr Nielsen. 1966; Englander et al.; 1972: Rosa & Richards. 1979). LVith the use of (‘OSY. quantitative exchange rates were obtained for 34 residues, and for four additional residues quantitative data could have been obtained by extension of the duration of the experiment (Table 1). For 14 additional residues, a lower limit for /c,(36”C) was established. Since this limiting rate is only approximately ten times slower than the average exchange rate for exposed amide protons in random coil model peptides (Englander it crl., 1972: and see below), WC are quit’e confident that t,hese 14 prot,ons represent, t,he exchange brhaviour of solvent,-accessible amide protons located near t,hc molecular surface in the solut,ion conformation of the protein. Overall, the COSY peaks of 52 of the total of 53 backbone amide protons had thus been individually assigned and the exchangfa behaviour of these protons was characterized. In the present experiment,s, a quench method was applied: i.e. the sample temperature was lowered before the start of the n.m.r. measurement Bec>ausr of the relatively long recording times needed to obtain a (‘OSY spectrum with workable signal-to-noise ratio. this will quite generally be needed. In a quench experiment. t,he observed resonance intensity. I(f). where t is t,he time during which exchange took place before the reaction was quenched. can be writt,en:
I, is the initial peak intensity. Ic, the exchange rat,e constant of interest,. t,,, the rate constant under tht time used t,o record the spectrum and k,,, t,he exchange conditions that prevail during the recording of the spectrum. Equation (2) yields:
It is readily apparent from protons, with k,,,t,,, -=$ 1, the can be neglected. For the quantitative evaluation of k,
equation (3) that for t,he very slowly exchanging exchange during the recording of the COSY spectrum more rapidly exchanging protons, straightforward is possible when fresh samples are prepared for each
SI’RFACE .~
1
STRI’CTI’KE -
OF
BPTI
IN
3.55
SOLL’TIOX
I
2 0
0
Oo
0 0 P0 G12
A25
1:5 0
2 4 2 5i
0
0
4
5
9
8
7
WI (P.P.rn.1
Fit:. 5. Contour plot ofthe absolute value 500 MHz ‘H COSY spectrum of a @02 M solution of Hl’Tl in ‘H,O, p2H 35, at IWC. Immediately before the n.m.r. experiment, the protein was dissolved in ‘Hz0 at 4°C. The spectrum was recorded in 12 h. The digital resolution is 53 Hz/point. The same spectral region is shown as in Figs 1 and 2. The assignments are indicated for the cross-peaks that could not be observed in the experiments of Figs 2 and 3. The vertical noise bands on the right of the spectrum are tails of the strong aromat,ic signals in the adjacent spectral region.
measurement of I(t), and identical measuring times, t,,,, are used for all recordings of POSY spectra. In this case, the pre-exponent’ial factor in equation (3) is the same for all measurements and the quantitative evaluation of 12, is not affected by the length of the recording time, provided that for each measurement I(t) is X1,/5, which is the practical limit where the signal intensity can still be measured. With rapid quenching of both pH and temperature to the conditions of minimal exchange rate. and provided that the protein is not denatured under t,hese conditions, the COSY experiment can thus be used for amide proton exchange
3%
(:. N'A(:SER,
AXI)
K. Wi'THKI(‘H
st,udies over a wide range of experiment,al caondit.ions. The spectrum of Figure 5 demonstrates that well-resolved COSY spectra can br obtained at conditions that are near t)hose for minimal exchange rates (Englander rot 01.. 1972). The fastest exchange brt’ween protons of the protein and deuterons of the solvent *HZ0 that may be measured with the presently described USC of C’OSY at lO’(’ (Fig. 5) is approximately MO5 min- ’ One arrives at this value with the assumption t’hat for quant,itat)ive st)udies the peak intensity after 12 hours at 1WC’ should br 2 Z&. Following equation (2) of l3nglander r>t (11. (1972). the average exchange rate for solvent-exposed amide protons in model pe@idrs at 10 (’ and p2 H 35 is of the order of 0455 rnin ‘. WC thus know that solvent-exposed amide protons exchange too rapidly to be sevn in (‘OS\- experiments in 2H20. hut the gall between thr limiting observable value and t.hr random c-oil exAanpe rat.r is only of the order of IO. The lower limits of the exvhangv rates at 36 and 68“(‘. respec*tivt~ly. for the protons that exchange too rapidly to 1~ seen at 10°C’. NY&W obtained by ext,rapolation from 10°C to higher temperatures. This means that from a lower limit /c,(lO”C’) > ON!5 min-‘, lower lirnits at lO’(‘, at highrr tjemperatures. k,(BB”(‘) > 0.1 mill- ’ and /z,(SX”C’) > 5 mirC ‘. wt’rt‘ estimated lvith the use of equation (2) of Englandrr rt nl. (1972): i.e. it lvas assumed that the erlthalpy of actjivat,ion for these protons is not lower t,han that of sol~cnt-cxposecl protons in model pept’ides.
(‘omparrd to the classical amide proton exchange measurements. which usv ra.dioaotivc tracers or infrared spectroscopy (Hvidt K: Xi&en. 1966 ; Englander et al.. 1972: Rosa Kr Richards, 1979). n.m.r. has the advantage of heing able to provide quantitative excahange rates for individual amide prot,ons in specGfied locations along the polypeptide chain. As a consequence of the improved spectral resolution in C’OSY spectra as compared to c*onvent’ional unidimensional n.m .I’. much rnorr cxt,ensive set,s of individual exchange rat,es can be measured. In BPTT, a complete mapping of thP amid? proton exchange rates for the ent’ire polypeptide chain was t bus obtained. &An alt,crnativr. novel procedure to map amide proton exchange rates in protein uses neutron diffraction techniques (Kossiakoff. 1982). A map of qualitative exchange data was thus recently obtained for c:rystalline t.rypsin (Kossiakoff, 1982). Since neutron diffraction and 21) n.rr1.r. can provide nearly complete mappings of amide proton exchange rat,es in single crystals and in non-crystalline the combined ust~ of the t’wo methods with the same environments. respectirelg. protein should open an avenue for direct comparison of the molecular dynamics in the crystal and in solut,ion. From the available information (Kossiakoff. 1982). the neutron diffraction technique should be more readily applicable for bigger proteins t,han 21) n.rn.r., but it might not be practical for proteins of any size t,o obtain quantitative exchange rat’es from neutron diffraction experiments. COSY can provide quantitat,ive c,xchangr measurements under a variety of different experirnental conditions. which will probably be of considerable import,anve for invest~igations of mechanistic asprds of prot.on exchange (see below). (‘onsidering
SI.RF.A(‘E
S’1’RI’(‘1‘17RE
OF
R1’1’1
IS
SOI,17’I’IOS
35;
that the use of both techniques for prot,on exchange studies is just being introduced. it, would appear premature to evaluate further their respective potentialities. but already now there appears t,o be a good chance that they could in many wa.vs provide complementary data on details of internal dynamics of proteins tha,t ww hitherto not amenable to experiment’al investigations.
The availability of complet,e sets of individually assigned amide proton exchange rates measured wit’h different conditions of pH and temperature should in the future help to clarify some uncertainties concerning the mechanisms 1)~ which t,he proton exchange reactions in BPTT proceed (Hilton & IVoodward, 1978.1979: LYagner & Wiithrich. 1979; Wtithrich d nl.. 1980n,b: Hilton et nl.. 1981). Here, n-e would like to describe a preliminary observation that might’ have some bearing on our understanding of exchange mechanisms. In Figure 6. the individual amide proton exchange rates for BPTI at 68°C’ and pH 3.5 are presented together with the intrinsic exchange rates. The latter are exchange rat’es for t’he random coil form of t,he polyprptide chain, which manifest the s~~~uen~~e cffert’s on the exchange of solvent-acc,essiI,le protons. Intrinsic rate constants f’or BPTI at 68°C’ and p2H 35 were calculat~ed wit,h the rules of Molda? rt nl. (197%). Figure 6 clearly shows that there is no simple relation between the two sets of rate constants and it can in most in&am-es be excluded that t,he relative values of’ thr exchange rates, k,, of neighbowing residues in the amino acid sequence are determined by seqnrnc~e effthcts. On the other hand. in an overall procws dominated by exchange bctwwn denatured prot’ein and the solvent one would intnit,ivcly expect the relative rates for individual neighboring protons to be
358
(:.
W$GNER
AND
K.
W~THRl(‘H
dominated by the intrinsic exchange rates. On this basis, the observations in Figure 6 might be forwarded as evidence against an exchange mechanism dominated by admixture of denatured protein molecules to the ensemble of BPTI conformers present in aqueous solution at 68°C (Hilton et al., 1981). (d) Crystal
structure
of
RPTI
and
amide
proton
exchange
in
solution
Figure 7 presents the accessible surface areas (Lee & Richards, 1971) for the backbone amide groups in the BPTI crystal structure (Deisenhofer & Steigemann, 1975) and the proton exchange rates in solution at two different temperatures. Overall, the qualitative similarity of the patterns obtained in the three graphs representing the accessible surface areas, k,(36”C) and k,(SS”C) along the sequence is quite striking. If one takes the pat!terns of exchange rates as an empirical manifestation of the three-dimensional protein structure, then the close similarity between the data obtained at 36°C and 68°C would indicate that the exchange at these two temperatures is from closely similar conformations. It would thus appear that Figure 7 provides additional evidence against the hypothesis that amide proton exchange at higher temperature is dominated by exchange from the denatured protein (Hilton et al., 1981). Some intriguing observations result from evaluation of the exchange rates in the light of the hydrogen-bonding network in the crystal structure. The conclusion that the exchange in the p-sheet is faster at both ends than in the central region: which resulted previously from a limited number of individually assigned exchange rates, is confirmed by the present, complet,e dat,a set. The most slowly exchanging protons are those of residues 21 to 24, which form the central strand in a short region of triple-stranded p-sheet (Deisenhofer bt. Steigemann, 1975). Except for Ala16, the exchange rates at 36°C of all other hydrogen-bonded amide protons in the p-sheet tend to be slower than t,hose for the hydrogen-bonded protons in the n-helix. The individual protons in the n-helix have also somewhat different, rates. whereby the exchange is slowest for Cys55 and Met52. Quite possibly. this is an effect of the disulphide bonds formed by Cys51 and (‘~~5.5. Further. it seems worth not,ing that the amide protons of TyrlO, Cysl4, Cys38 and Lys41, which in the crystal structure are hydrogen-bonded with internal water molecules (Fig. 7: Deisenhofer & Steigemann, 1975), do not, have unusual exchange rates. As one might have anticipated from the vanishing or very small (Lys41) static accessible surface areas for these residues in the crystal structure. we observed exchange rates k,(36”C) < 0.1 min-’ (Table 1 : Fig. 7). The exchange studies of amide protons that are located near the protein surface in the single crystal structure should be a useful tool to probe the surface of the solution conformation. One might, in a preliminary evaluation for example, assume that all the amide groups that have a non-vanishing accessible surface area, and are hence in van der Waals’ contact with the solvent (Lee &, Richards, 1971), should exchange rapidly; i.c. with &,(36”(Y) > 0.1 min-‘. k,(36”C) values would accordingly correspond to groups with zero accessible surface 10.1 min-’ area. Figure 7 and Table 1 reveal that there is a single residue that has zero accessible surface area and k,(36”C) > 0.1 min - ’ : i.e. Glu49. If one assumes that
SI’KFAC’E
i lo-2/ 4 10-j
10.6.
L
STKI-(‘TITKK
OF
HI’TI
IN
SC)l,lTTION
360
G. WAGNER,
.4X1)
K.
Wi’THKlCH
the protein surface is dynamic in the sense that the atoms undergo fluctuations about an equilibrium position, it is quite conceivable that’ the solvent exposure is increased as compared to the static accessibility in the crystal st,ructure (Lee 6: Richards, 1971). On the other hand, there are ten residues that would in the crystal structure be in van der Waals’ contact wit,h the solvent but, have in solut,ion. Theke are Asp3. Glu7. Gly12, Alal6. Ala25. k,(36”C) I 0.1 mind1 Gly28, Cys30, Thr32, Va134 and Lys41. Here it appears intuitively rather unlikely that dynamic fluctuations of the protein structure could reduce the soIlvent accessibility as compared to the static accessibility in the crystal, and there is an indication that these residues are in different environments in the cryst,al and in solution The solvent-accessible surface area for the crystal structure (Table 1) was computed for an isolated BPTI molecule. Hence, lack of correlation with the amide proton exchange rates in solution could be due either to intermolecular aggregation at the high protein concentrations for which t,he data in Table 1 and Figure 7 were obtained, or to rearrangement, of the protein surface structure between the crystal and t’he solution. Further studies of the apparent, discrepancies between the conformations of BPTI in single crystals and in solution are in progress. These will include comparison of the proton exchange data in Table 1 with corresponding measurements of exchange rates in very dilute solutions of BPTI, to distinguish between influences of intermolecular aggregation and conformational changes in monomeric BPTI. We envisage that the observation of apparent, discarepancies between the structures of the protein surfaces in the c=rystal and in solution by amide proton exchange studies could be a start,ing point for site-specific use of different, more laborious n.m.r. experiments capable of providing direct information on the surface conformation in these locations and/or on struc*tural details of intermolecular aggregation in concentrated protein solutions. We thank the Schweizerische Nationalfonds (project 3.538.7!)) for financial support. the Fnrhenfahriken Bayer A(: for a generous gift of Bl’TI (T rasylol”), I)r P. (‘hothia for a complete listing of accessible surface areas in KI’TI. and ,Mrs E. Huber and Xlrs E:. H. Hunziker for the careful preparation of the manuwript and the illustrations.
REFERENCES Allerhand. A.. Doddrell, I>.. (Glushko. V.. C’trchra.n. 1). W.. Wenkrrt. E.. Lawson. I’. J. & (Kurd. F. R. N. (1971). J. ;1 vtw. Phpm, Sot. 93. .544-;546. Anil Kumar, Wagner. G.. Ernst, R. R. Rr Wiithrich. K. (1980). Biockem. Biophys. Kes. Common. 96. 1156-1163. Arseniev, A. S., Wider, G.. Joubert. P. J. & Wiithrich, K. (1982). .J. Mol. Bid. 159, 323-352. Aue, W. I’., Bartholdi, E. & Ernst. R. R. (1976). .I. Chem. Phys. 64, 2229-2246. Baumann, R.. Wider, (i.. Ernst. R. R. & Wiithrich. K. (1981). J. Magrt. Resun,. 44. 408-406. Billeter, M., Braun, W. & Wiithrich, K. (1982). .J. Mol. Biol. 155, 321-346. Bundi. A. 8r Wiithrich. K. (1979). Riopolyrr~rrs, 18, 285-298. Chothia. (1, & Jnnin. ,J. (1975). iV~~t?~w (Londotr). 256. 705-708. Deisenhofer. J. & Steigemann. W. (1975). =tctn C’rystnllog~. sect. H. 31. 238-250. Englander, S. W. & Paulsen. A. (1969). Riopol~~tner.~. 7. 379-393.
SlTKFA(‘E
STRV(“l’L’HE
OF
HI’TI
IS
SOI,I’TIO?1’
36 I
ICnglandrr. S. W.. Downer. N. W. & Teitell)aum. H. (1972). ilnncr. Kw. Hiochrw. 41. 903 !W. Hilt,on. B. I). 8 Woodward. C. K. (1978). Hiochrmistry. 7. 332.5-3332. Hilt,on. W. I). Xr Woodward. C’. K. (1979). Biochu~ristry. 18. 5834~5,844. Hilton. H. I)., Trudeau, K. & Woodward. C’. K. (1981). Rioch~~zi.stry, 20. 4679-4703. Huber. R. (1979). Trends Biochem. Sri. 4. 227-230. Hridt. A\. 8: Sielsen, 8. 0. (1966). ildvnn. I’rotrin C’hrm. 21, 287-386. Kalilrichenko, I’. (1976). Stud. Biophys. 58. 2X-240. Karplus. 11. & hlcCammon. J. 4. (1981). (I.I1.C. CM. Rrr?. Riochrrrc. 9, 293-349. Kossiakoff. A. A (1982). Saturr (Lor&)rr). 296. 713-721. I,w. H. Kr Richards. F. 31. (1971 ). J. Mol. Rio/. 55. 379-400. Molday, R. S.. Englander. R. W. & Kallen. R. (:. (1972). Biochemistry. 11. 150-158. Nagayama. K.. Hachmann. I’.. Wiit)hrirh. K PL Ernst. FL R. (1978). ,/. Magn. Rvson. 31. 133 148. Xagayama, K.. b’iithrich. K. & Ernst. R. R. (1!179). Rioch~wn. Hiophys. Krs. Con~rnu~. 90. 305
3I 1
Sagayama. K.. Anil Kumar. WLithrich. K. & Ernst. R. R. (1980). .J. Magn. Rrson. 40. 321 334. l’erutz. M. (1970). ,Vnture (Londort). 228. 726-739. Quiocho. F. -4. & Lipscomb, W. K. (1971). .3dvnn. Protein Chenr. 25. l-59. Ribriro. A. :I.. King. R.. Restivo. C. Nr Jardetzky. 0. (1980). .1. =In/w. Ph~m. Sot. 102, 404(t 1051. Richardson. .J. S. (1981). ddzar/. I’rotrin Chrrn. 34. 167-339. Richarz, R.. Sehr. P., Wagner. G. 8r Wiithrich, K. (1979). J. Mol. Biol. 130, 19-30. Ricaharz. It.. Nagayama, K. & Wiithrich. K. (1980). Riochemistry. 19. 5189-5196. Rosa. J. ?J. & Richards, F. M1. (1979). J. Mol. Rio/. 133. 399-416. Saunders. >I. 8: Wishnia. A. (19%). =I r/n. S. Y. dcnd. Ski. 70. 87&874. Scheinblatt. M. & Rahamin. Y. (1976). Riopolymers. 15, 1643Gl653. Schulz. (:. E. 8: Schirmer, R. H. (1979). I’rinciplas of Pro&in Stmctuw. Springer, New York. Wagner. (:. & Wiithrich. K. (1979). .J. No/. Rio/. 134, 75-94. Wagner. (:. & Wiithrich. K. (1982). J. Mol. Hiol. 155. 3477366. Wagner. (i.. De Marco. A. & Wiithrich. K. (1976). Biophys. A’twc/. Mech. 2. 13%158. Wagner. (i.. Anil Kumar & Wiithrich. K. (1981). Eur. J. Biochem. 114. 375-384. Wider. (i.. Raumann. R.. Nagayama. Ii.. Ernst. R. R. & Wiithrich. K. (1981). .J. Mogw. Reaon 42. 73-87. Wider. (i.. Lee. H. K. & Wiithrich. K. (1982). .J. Mol. Biol. 155. 367.-388. Wiithrich. K. (1976). AVMR in Riologicol Re,search: Ppptides and I’rokins, North-Holland. i\msterdam. LViithrich. K. R- Wagner, (:. (1979). J. MO/. Biol. 130, I-18. Wiithrich, K.. Hug&r, A. & Wagner. (:. (19800). J. Mol. Biol. 144, 601-604. Wiithricah. K.. Wagner, G.. Richarz. R. B Hraun. W. (19806). Biophys. J. 10. 549p560. LViithrich. K.. LVider. G.. Wagner. G. Kr Braun. W. (1982). J. Mol. Rio/. 155. 31 l-319. Edited
by
I’. Luzzati
Amide
160, 343-361
Proton Exchange and Surface Conformation Basic Pancreatic Trypsin Inhibitor in Solution
Studies with Two-dimensional
(Received
Nuclear
11 February
Magnetic
of the
Resonance
1982)
A norc~l approach for studies of amide proton exchange in proteins is presented. It relies on measurements of the amide prot,owC” proton cross-peak intensities in the two-dimensional homonuclear correlated ‘H nuclear magnetic resonance spertra. The protein is dissolved in ‘Hz0 and the solution is exposed to the conditions of p2H and t,empwature where the exchange rates are to he measured. After variable int,ervals. the amide proton exchange in a sample of this protein solution is quenched by lowering the temperature and possihl,v hg p2H variation, and a C’OSYt spectrmn of this sample is then recorded. Comparison of the NH-C”H cross-peak int,rnsities in t,he specka recorded after different exchange t,imes yields exchange rates for the individual amide protons. The main advantage compared to previously described techniques is that a much more complete set of individual arnidr proton exchange rates can he ohtjained. In the hasir pancreatic trypsin inhihitor. where all the amide proton rcsonanccs were previously individualI> assigned. quantitative exchange rates were obtained for 38 of the total of 53 hac*kl)one amide protons. and for I? addit,ional protons lower limits for the exchaltge rates lverr estahlished from comparison of the COSY spectra recorded in H,O and in 2H20. Proton exchange data lvere thus for the tirst time obtained for numerous peptide groups t,hat are located near the prot,ein surface in the sin&~ crystal struc%ure of RI’TT. For sonle locations on the protein surface. it appears that the amide prot,on exchange rates cannot he cwrrrlated readily wit,h the static, awrssihle surface areas in the cr,vstal structure.
1. Introduction stability of biologically active spatial protein structures relies on a complex interplay of a multitude of weak, non-bonding interactions among different atoms of the polypeptide chains and between the polppeptide chain and the surrounding medium. The lat)ter may, for example, be an aqueous solvent, an ordered lipid matrix The
344
(:. b'A(:NEK
ASI)
K. Wi;'FHRICiH
in biological membranes, or the ordered aggregation in single crystals used for Xray studies. Since the contribution of each individual non-bonding interaction to the free energy that stabilizes the protein conformation is typically of the same order of magnitude as the thermal energy at temperatures near 300 K, these “secondary bonds” are constantly broken and reformed. As a, result, protein molecules are highly dynamic structures. On the one hand, since they depend on intramolecular interactSions and on interact,ions wit,h their environment. protein conformations adapt readily to local changes of the polypeptide covalent structure and to changes of their surroundings. Examples are the conformational transition from trypsinogen to trypsin (Huber. 1979). t,he local conformation change in carboxypeptidase A upon substrate binding (Quiocho & Lipscomb. 1971). the quaternary structure transitions in tetramerir hemoglobin (Perutz, 1970) and quite generally the potential adaptability built into multi-domain protein structures with flexible hinge regions (Huber. 1979: Schulz Br Schirmer, 1979: Richardson. 1981). On the other hand. protein mole:cules in thermodynamic equilibrium situations undergo time fluctuations about, an average set, of atom co-ordinates. These st,ruct,ure fluctuations cover a wide range of frequencies. amplitjudes and energies of activation. They have been observed, for example. in nuclear spin relaxation measurements (Allerhand rt al., 1971 ; Ribeiro et nl., 1980; Richarz et al., 1980), aromatic ring flips (Wagner et ul., 1976) and amide proton exchange studies (Hvidt & Nielsen, 1966: Englander et al., 1972: Richarz et al., 1979), and were extensively investigated by molecular dynamics calculations (Karplus Qi McCammon, 1980). This paper describes amide proton exchange measurements wit,h the use of two-dimensional nuclear magneticresonance. The results of these experiments bear on both the adaptation of the molecular conformation to changes of the protein environment and the time fluctuations of the protein about an equilibrium set of atomic co-ordinates. In the above-mentioned, well-documented examples of conformational changes upon variation of the protein covalent’ structure or the protein environment, the initial and final states of the protein could both be studied by X-ray methods in single crystals (Perutz, 1970: Quiocho & Lipscomb. 1971 : Huber, 1979). Different experimental techniques must, be applied t,o investigate how the molecular conformation adapts to the change of environment, when a protein is transferred from single crystals to a non-crystalline state. Such a technique should be capable of determining the polypeptide conformation with comparable detail to t.hat of the single crystal X-ray structures. We have recently proposed that this cbould be achieved with ‘H r1.m.r.t experiment,s (Wiithrich of ai.. 1982), and as a first, fundamental step complete individual assignments werp obtained for protein ’H r1.m.r. spectra (Rilleter rt al.. 1982: Wagner & Wiithrich. 1982: Wider et al., 1982: -4rseniev et al., 1982). In this paper. 2D n.m.r. is used to investigate the exchange with the solvent of all the backbone amide protons in the basic pancreatic trypsin inhibitor. Combined with the previously obtained resonance assignments. these data serve t,o probe the molecular surface of KPTI in solution. which will then be correlat,ed with data on the crystal structure. t See footrlotr to p 343
sITRFACE
STRI-CTlTRE
OF
BPTI
IN
:Lki
SOLl”~lON
Amide proton exchange measurements have long been applied for studies of internal fluctuations in proteins (for reviews, see e.g. Hvidt & Nielsen, 1966: Englander et al., 1972), and already in 1958 Saunders & Wishnia demonstrated that proton exchange between proteins and solvent 2H20 can be observed by n.m.r. While other techniques for kinetic studies of specific groups of protons have been described (e.g. Rosa & Richards, 1979), the potentialities of n.m.r. for measurements of individual amide proton exchange rates appear to be quite unique (Wtithrich. 1976). With the experiments described in this paper, one obtains for thca first time a complete data set, i.e. the individual exchange behaviour ten be charact,erized for all the backbone amide protons in a protein. This should be of great value with regard to further clarification of the mechanistic aspects of amide proton exchange from globular proteins (see e.g. Wagner & Wiithrich, 1979: Hilton et al., 1981), and one can hope to obtain a more complete view of the internal “breathing modes” (Hvidt & Nielsen. 1966) than has hitherto been possible.
2. Materials and Methods Basic pancreatic
trypsin
inhibitor
(Trasylol Cm , Havrr Levrrkusrn)
was obtained
from the
Farbenfabriken Bayer AU. The solutions used for the n.m.r. recordings contained 0.02 MHI’TT and the pH was adjusted by the addition of minute amounts of HCl and NaOH. whereby in the 2H20 solution the pH meter readings were used without correction for isotope effects (Kalinichenko, 1976; Bundi & Wiithrich, 1979). For the experiment in Fig. I. a mixed solvent of 90% Hz0 and 10qb ‘HzO. pH 4%. was used. so that all the backbones amide proton resonances were present in the spectrum. To observe a maximum number of it is important that from t,he first contact amide protons in a protein solution in ‘H20. between protein and ‘H,O the p*H IS near the p2H minimum for proton exchange (Wiithrich &r Wagner. 1979). Therefore, to prepare ‘H,O solutions of BPTl for the proton exchangra measurements. the protein was first dissolved in H,O and the pH adjusted to 3.5. h’ext. RPTT was Iyophilized and dissolved at 24°C in 2H20. The resulting p2H of the RPTT solution wa,s in all experiments in the range 3.5 k 0.2. \\,hich was then adjusted to 35 with the use of a combination glass electrode. The measurements in this paper were all done with homonuclear 2-dimensional (21)) correlated spectroscopy (COST) (Aue et al.. 1976). COSTi uses t,he pulse sequence (Aue cjt rrl.. 1976; Nagayama et frl., 1980: Wagner P/ nl.. 1981): 190’ -t,
-90”-t,],.
(1)
where t, and t, are the evolution period and the observation period, respectively. To obtain a 21) n.m.r. spectrum, the measurement is repeated for a set of equidistant t, values. To improve the signal-to-noise ratio and to eliminate experimental artefacts. )L groups of 16 recordings with different phases were added for each value of 1, (Nagagama ~1 nl.. ISi!!. 1980). At the end of each recording. th(s system was allowed to reach equilibrium during a fixed delay of 1.2 s. The spect,ra were recorded at 500 MHz on a Bruker WM 500 spectrometer. The spectrum in Fig. I was obtained from 512 measurements. with t, values from 0 to 47 ms and 2048 points in t2. To reduce the observation time, the spectra in 2H20 were obt,ained from 2% measurements, with t, values from 0 to 24 ms and 1024 points in t,. To end up with a 1024 x 1024 point data matrix in the fmqucnc? domain. which corresponds to a digital resolution of 5.7 Hz/point, the time domain matrix was in all experiments expanded t,o 2048 points in t, and 4096 points in t, by “zero-filling”. Quadrature detection was used for detection of the individual free induction decays, with the carrier frequency at the low-field rnd of the spectrum. For the measurement in H,O. the
346
C:. LZ’A(:NER
ANI)
Ii. b\‘i”PHKIC:H
solvent resonance was suppressed by select.ive. continuous irradiation at, all times rxwpt during data acquisition (t2 : Anil Kumar rt nl., 1980). Prior to Fourier transformation. thr time domain data matrix was multiplied in the t, dirt&on with a phase-shifted sine bell. sin (n(t+t,)/t,), and in the t, direction wit,h a phasr-shifted sine-squared bell. sin’ (~(t c/,)/t,). The length of the window func~tions. t,. was adjusted for the hells to reach zero at the last experimental data point in the t, or t, direction. respwtively. The phase shifts. t,if,, were l/32 and l/64 in the t, and t, direction. respectively. The Hz0 spectrum in Fig. 1 \vas further improved by symmetrization (Raumann PI trl.. I!)81 ). The absolute value presentation was used in all the experiments. Quantitative measurements of the amide proton clxchxnge rat’es were made at 36’ C and 68°C’. The *H,O solutions of BI’TT used for t,hrsc> t~xp(~riments were prepared at 24’C. Immediately after the protein ha,d been dissolved alrd the p2H adjusted to 3.5. t,he solution was heated to the desired exchange temperat.ure for a certain length of time. Then it MBS cooled to 24°C’. and the C”OXY spectrum was rewrded during 11 h at 24°C. A new sample \~as prepared for each of the measurements with rxrhangtx time-s at 36°C’ of 0. 10. 240. 660 and 1720 min (Fig. 3). rlfter the completion of the n.m.r. rwording. the sample of the 1720 min experiment was further used for all t,ho remaining measurements with longw exchange times (see Fig. 3), where the excahangt, during the 11.mr. rerording at 24 C is Ilegligibly small compared to the exchange during the kvaiting timths at 36°C’. Similarly. IIe\v samples were prepared for the individual measurements at 68-C’. \vhicah wer(’ recorded after uaitillg t#imrs of 0, 5. 10 and 15 min. The same sample as for tht, I,5 min expc~riment, was further used for 2 additional measurements lvith exchange times of 60 and 120 min. To measure resonance intensities. cross-wctions through the COS\’ spectra wre plotted (Nagayama rt al.. 1978) and the height of the individual peaks was measured. For all the measurements. the spectra \vrre recorded with identical instrument settings alld the datta were handled in identical \vays. The peak heights ilr each spectrum were measured relatiw to t,hose of the well-known and well-separated low-field C’H-C”H cwss-peaks of Tyr2l and Cys30 (Wagner & Wiithrich. 1982). Since wit,hw C” nor (‘” protons exc*hange wit.h *HzC). these two peaks have intensity I in each spec%rum independent, of the exchange time and exchange temperat,ure. The rate constants in Tablr 1 were obtairrcatl from a nolr-linear least-squaws tit of the c~xperimental data to an c~xponential function. ;2n error analysis was performed for all those protons. for which the time dependence of the peak illtensity could be measured at 4 or more points. For 19 amino acid residues in KI’TI. the amide proton rxchallgc is so fast that their NHC”H COSY cross-peaks were absent, already frortl the spectrum recorded after the shortest exchange time used at 36°C. To obtain furt,hcr information OII these exchange reactions. new KPTI solutions in ‘H,O were prepawd at 4’(’ arId the p2H n-as again adjusted to 3.5. .L\ COSY spectrum of one of t,hesc samples \vas rw~ordrd iI1 12 h at 1O’C immediateI?- after sample preparat.ion. The seco~ld sa.mple \vas heated to 3BC‘ for 10 min twfort- a (Y)S\. r(wjnance assiglunerits at spectrum was recorded in 12 h at lO,C. For t.hrsr experiments. 10°C were obtained with th(* previously dcs~rilwd t.whniques (Wagner 8: Wiit.hriqh. 194’2).
3. Results In two-dimensional correlated spect,rosc*opy. one obtains a square array of resonance peaks in the w1-w2 plane (Fig. I ), In the complete COSY spectrum, the to the normal, one-dimensional so-called diagonal peaks with w1 = w2 correspond spectrum (see e.g. Nagayama et al., 1980; Wagner & Wiithrich, 1982) (the diagonal peaks are not shown in any of the Figures in this paper). Any two diagonal peaks at w1 = w2 = wA and w1 = w2 = wx that originate from protons linked by scalar spinspin coupling (J-coupling), are connected by a pair of “cross-peaks” located in symmetrical positions with respect t.o the diagonal at (wl = wA, w2 = wx) and
SI’RFACE
STR[lCTURE
OF
BPTI
IN
347
SOl,L-TION
w2 (p.p.d
r-
8 I
9 I
IO I
7 I
COSY Hz0 80 oc
3
6
I 9
I 8
I 7
-6
WE (p.p.m.)
FIN:. I. Symmetrized (Baumann et al., 1981). absolute value 500 MHz ‘H COSY spectrum of a 002 VI solution of RPTT in a mixed solvent of 90(X Hz0 and loo/* *H#. pH 46. at 80°C. The spectrum was recorded in approx. 24 h, the digital resolution is 5.3 Hz/point. The entire spectrum was presented previously (Wagner & Wiithrich, 1982). Here. the region (wl = 1~5t06~0p.p.m..w2 =6~6tolO%p.p.m.). is shown in a stacked plot representation in the lower part of the Figure, which affords a “3 dimensional” view of the spectrum. and as a contour plot in the upper part. The temperature of 80”(’ was chosen to avoid the appearance of cross-peaks with the labile protons of arginine and Iysine side chains. which show up at lower temperature at this pH value. The spectrum thus contains only NH-C”H cross-peaks. whereby all the amino acid residues can be observed except Argl. Gly37 a,nd the 4 prolinc residues (Wagner & Wiithrich. 1982).
348
(:.
\Y.i(:NEH
ASI)
IC
lVi’THKI(‘H
one works with a mac~romole~ule sucsh as a protein. a ( Wl = wx> w2 = wA). When single (‘OSY spect,rum can provide information on all t,he proton-proton fJconne&ivities in t,he molecular structure. The presentj applic*ation of (‘OSY for studies of amide proton rxchangt, rates relies on measurrments of’ the time dependence of the intensities of the pofylwptide harkbone amide I)roto~~A” proton cross-peaks. which we ha,ve previously c+alft~l the “n.m.r. fingerprint of the protein Aructurr” (Wagner & iViithric>h. 1982). In a protein C’OSY spwt’rum rrwrded in H,O at pH near 3.5. each amino acid residue gives rise to one SH- (‘“H cross-peak. Exceptions are the gfycine residues. which give in general two cross-peaks. t,he N~tt~rminal residue. where t,hr amino protons usually exchange too rapidly t)o lw ohserved (Scheinblatt Cy: Rahamin. 1076: Bundi R- Wiit’hrich. 197!1) and. of course. the prolinc residues (LVagncr & Wiithrich, 1982). The TL’H-C”H cross-peaks are usually all cwnt,ained lvithin a limited spect)ral region, which contains no 01’ rt3r.y fbw othrr cmss-peaks, tlt~pending on the conditions of the experiment. L2’hcn the arnino acid sequenw is known. inspection of t’his region of t,h(L (‘OSY spectrum thus pro\-ides a rapid clheck of whether all or most of the amino acid residws c.an lw observed and their resonances resol\-ed in the 21) n.rn.r. spectra. For KPTI _ all N HP(‘“H cwss-peaks are located in the spectral region (wl = 1.8 to 5.8 p,p.m.? w2 = 6.7 to 10.5 p,p.m.). and at the pH and t’emperature of the experiment in Figure l this region contains no ot’her crosspeaks (Wagner & LViithrich. I!X%Z)j-. Figure 1 presents an almost wmplrte The NH-(‘“H cross-pwks are fingerprint of t,he BPTI amino acid sequcnw. resolved for 52 rrsidurs. i.e. all rxcrptj Ar’gl , (:lyX and t,he four profine residues. and all the peaks were previously assigned t,o specific residues in the amino acid sequence (N’agner B Wiithrich. 1982). X reduced fingwprint of the protein strwturc is afforded hy the same region of a (‘OSY spectrum recorded in 2H20 (Fig. 2). SHY (‘“H (aross-peaks are seen only fol those residues for which the amide proton exchanges rela,tively slowly \vit,h 2H of the solvent. Inspection of Figure 2 reveals that instead of the cross-peaks for 52 residues seen in Figure 1, only 33 residues are manifested. The peaks of these 33 residurs are identified in Figure 3. Individual assignments for all the backbone arnide prot,ons of BPTI have been described at 68°C and pH 4.6 (Wagner C% LViithrich, 198%). For all the protons observed in 2H20, the assignments at 24°C’ and p2H 3.5 were independently established with the same techniques (some assignmen& at 21°C’ are documented 1)~ \Vagner it crl., 1981). (‘ompared tjo
up (p.p.m.)
COSY 24 T
7 I
8 I
9 I ‘H,O
;P
6
I 9
I 6
1 7
w2 (P.P.~)
FIG:. %. Ahsolute value 500 MHz ‘H POSY spectrum of a @02 M solution of KI’TI in ‘H,O. pZH 3.5 at 24 (‘. Irnmrdistety twfore the n.m.r. experiment. the protein was dissolved in ‘Hz0 at 24 (‘. Thcs solution vxs thrn kept at 36’C for 10 min and then cooled again to 24’C. at which trmperature the spectrum was recorded in 12 h. The digital resolution is 53 Hz/point The same spectral rqion is show11 as in Fig I, Only the NH-C”H cross-peaks with the stowty exchanging amide protons (Wiithrich k The vertical noise bands lwtwrrtr 6% and \Vagnrr. 1079: Wagner & Wiithrirh. 198%) are observed. i.5 p,p.m are “tails” of the strong aromatic signals in thv adjacent spwtral wgion.
(:.
I+‘A(:NER
AN11
K.
Wi;THRlCH
Y23
“‘O
N24
0 F45
min
1720
mm
660
240
mln
0
T32
0
A27
-
I’
B
IO mm
, IO
9
0 c30
, 8
i
I 7 W* (pwn.)
FIN:. 3. Absolute value 500 MHz ‘H COSY spectra of 0.02 M solutions of BPTI in *HZ0 recorded at different times after the protein was dissolved. The solutions were freshly prepared at 24°C and then kept at 36°C to allow exchange of protein amide protons with *H of the solvent. At the times indicated in the Figure, a particular solution was cooled to 24°C’ and a spectrum was recorded in 12 h. The digital resolution is 5.3 Hz/point. Compared to Fig. 2. the size of the plot was reduced to the region (wl = 3.6 to 60 p.p.m., (02 = 66 to 10.8 p.p.m.), cutting off the cross-peak of Cys51 at (wr = 1.85. w2 = 7.05 p.p.m,) and 1 of the 2 cross-peaks of Gly36 at ( w1 = 3.4, w2 = 8.6 p.p.m.). Furthermore, the vertical noise bands between 66 and 7.5 p.p.m. have been covered with white paint. The peaks that disappeared in the course of the experiment are identified in the last spectrum. where they can be observed readily. Thus this Figure affords a qualitative survey of the exchange rates (for quantitative data. see Fig. 4 and Table 1). The peaks that did not disappear within 80,000min are identified in the last spectrum. The disappearance of the C51 cross-peak, which is outside the spectral region shown. is indicated with an arrow in the spectrum taken after 5880 min.
w2 = 6.23
p.p.m.
116 G26
1
I
I
r
---. 7
J I
6
5
4
3
6
5
4
r
3
I
I
7
-I
3
6
5
4
3
6
w,
5
1
I
4
3
(rw0.m.)
I+:. 4. Vrrtioai c.ross-sert~ions of the spectra shown in Fig. 3 taken at 1 different positions along thr (u2 axis. At thr top of the Figure. those residues are idwtified for which w2 coincides with w2 ofthr crosssection The cross-peaks of the residues indicated in parentheses are located so close to the c-ross-sections that tails of the peaks are observed in thvsr presentations.
Figure I. t,hr cross-peaks in Figures 2 and 3 are considerably broader. This is a consequence of both the lower temperature and the smaller number of t, and l2 values used to record the spectra (see Materials and Methods). For the 33 residues that can be observed in Figure 2, the time-course of the crosspeak intensity was followed over a period of approximately two months (Fig. 3). For the quantitative measurements, cross-sections of the COSY spectra (Nsgayama d (11.. 1978) were used as illustrated in Figure 4. It seems worthwhile t,o point out the excellent signal-to-noise ratio achieved with the present experiments (Fig. 4) and to emphasize that the spectral analysis relied on measurements of the peak heights relative to peaks t,hat are known not, to vary with time (see Materials and Methods). For four residues, only upper limits for the exchange rates at 36°C’ were obtained (Table I), since the peak intensities were essentially unchanged after 83040 minutes (Fig. 3). For 26 residues, t’he time change of the peak intensity could be followed in four or more of the spectra in Figure 3. For these, error limits for k, (36°C) ranging from +3 “/b to _+ lo’?&, depending on the proton, were computed (Table 1). For the remaining three residues, less than four data points were obtained. so that error limits for k, could not be established reliably (Table 1).
352
(:.
WAGNER
ANI) T.4Irl,l~;
Rate constants
Amino acid residue
\V~THKICH
1
10-” ,min-’ , + loo/,) for thr exchange oj’ i~~divid~ual protons in BPTI at p2H $5 and 36°C and &I”(
k,(in
backbonr
amidr
k,(36Yyt
Argl Asp3 Phe4 CyvsT, I,eu6
n.o 10 PHI 0.3 1 023 I2
GlU7
TyrlO Thrl I Gly12 Cysl4 LyslS Ala16 Argl7 Ilel8 lIeI Arg20 T+2l Phe22 T)T23 Am24 Ala25 Lys26 Ala27 Gly28 Leu29 (‘\40 Gin31 Thr32 l’he33 Val34 T)Y% Gly36 my37 f’ys3X Arg3’) Ala40 Lysll Arg4P Am43 Am44 Phe45 Lys4ci Ser47 Ala48 Glu49 Asp50 Q-&51 Met52
Ii.
-100
* 1.1
23: f
,.,;
87
f 00022 < OAO4
-100 -4, tr ’ I3
091 1 - 80 0aoO48 43
om1o 3.1 MO44 (FOTO 11.0. - 80
f 0.5: - 100 om75 04013
f -100 f -100
II.0. - 330 f 93 43 14 93 f
f 032 0,057
f 65 f 7.1 56 5.9 62 12 f - 32:)’ 52 9% f
11 210 ti+?i 140 I0 50 n.o. f t 1’ 180 f f 3i 20 f f f f - 5:; II
37.000 51 .OOO 1 I0.ooo <56,000 39,000 %4.000 39.000 40,000 88.000 69.000 .56.000 35.000 36.000 30,000 35.000 44 .ooo 28.000 28.000 88.000 78,000 35,000 .56 000 8o:ooo 59.000 88.000 78.004~ I 23 .ooo 44.000 36 .ooo 24.000 99 .Ooo 16R.000 li5.000 69.000 .i6,000 35.000 70.000 I 39 .ooo 196.ooo 62.000 37,000
174,000 5N ,000 99.000 i2.000 144.000 56 .ooo
+ + + +
+ + +
B
i
B
+ +
a
+ +
+
+ + +
4
SITRF.4(‘E
STRI’(‘T1’RE
OF
BPTI
IX
SOLI’TIOS
xi3
TAHLE 1 (continued In In solution k,(36”(‘)t 0.07 1 0~40 0~036 2.1 f f
Accessible surface area$
k,(68”r)t 16 33 20 79 f f
3.5 .ooo 174.000 174,000 156 . 000 158.OOO 63.000
04 0.0 04 0.0 3.0 11.7
the
crystal
Hydrogen bonding + + + +
structure
)I
Regular secondary strurture
7
\ 1 \ 1
For ti8 (’ and p*H 3.5 the intrinsic exchange rates, k,,,, are also given. Further. the following properties of the individual amide protons in the crystal structure of BPTT are listed. Accessible surface area, hydrogen Ironding and. where applicable. location in a polgprptide segment which forms regular secondary structure. I- The numtwrs give k, in IV3 min - 1 k IO’&. The sign - in front of a number indicates that less than 4 experimental points were measured and therefore no error analysis was warranted. n.o.. not ohserved. Indicates that the NH resonance of this residue could not be detected in the COSY spectrum. f indicates that the amide proton exchanged too rapidly to he seen in the POSY spectrum recorded at 10°C (see the text). ‘I’hv following limits for the exchange rates of these protons were estimated: k,(36”C) > 0.t min-’ : k,(68’(‘) > 5 min-’ (see the text). : Intrinsic rate constants at p2H 3.5. which reflect inductive effects of neighbowing residues. \vew valwlated according to the rules of Molday rt cl/. (1972) starting from equation (2) of Englander rt crl. (1972). To obtain these data, pK, values of 3.4. 3%. 3.8. 3.0 and 29 were used for 4sp3. Glu7. Gtu49. Asp50 and thv C-terminal Ala58. resprctivel,v (Wiithrich & Wagner. 1979). k,,,,(BS”C) in IV3 min-‘. 5 Acwssihlr surface area as defined by Lee & Richards (1971). The calculations for BPTI are described in (‘hothia & .lanin (tR75). The data used here are taken from this reference and from a complete listing of t,tu, solvcrlt acwssihilities for all the atoms in BPTI, which was kindly given to us hy Dr C. Chothia. /I A + sign indicates that t,he amide proton of this residue was assigned to a hydrogen bond in the rrtinc~d (,rystal structure (Deisrnhofer & Steigemarm. 1975). * 1 and p, resprctivcl~.indicate that the amide proton was found t,o tw in a hydrogen bond that is part of an \-hc>lix or an antiparallel p-sheet in the refined crystal structure (Drisenhofer & Steigemann, 1975).
(‘orresponding experiments were done at 68°C’. The exchange at this temperature could be followed for 31 amide protons (Table 1). For 28 residues, k,(68”C) could be established wit,h error limits of + lOo/,,, and for three residues. approximate exchange rates without error limits were obtained. Addit’ional rxperiments were used to further discriminate among t,he 19 amide protons that, exchange too rapidly to be seen in the first spectrum recorded in the experiment of Figure 3. A 2H,0 solution of BPTI was prepared at, 4°C and immediat,ely thereafter a COSY spectrum was recorded at 10°C in 12 hours. Figure 5 shows that, compared to Figures 2 or 3, five additional residues could thus he observed. In a different experiment, where an identical sample was heated to 36°C’ for ten minutes before the COSY spectrum was recorded at IO”C, two of these ti1.e cross-peaks were too weak to be seen and the other three had lost much of their intensity. From these observations, we estimated that k,(36”C) - 0.1 min-’ for the five prot)ons identified in Figure 5. For the 14 prot,ons not seen in the experiment at IO’C’. we expect accordingly that k,(36”C) > 0.1 min-’ (Table 1). (See Discussion for furt,hrr details on how these values were obtained.)
3.54
(:. WA(:NER
ASI)
K. M'i'THRIC'H
4. Discussion (a) 21) TZ.m.~.
for
studies
of nmidr
proton
rxchanp
rates
The main advantage of the presently described use of C’OSY for studies of amide proton exchange in proteins is the improved spectral resolution, which allows observation of separate peaks for nearly all amide protons in RPTI. Compared to corresponding unidimensional n.m.r. experiments. the overlap of amide proton resonances with those of the aromatic protons is completely removed, and the resolution between different amide protons is great’ly improved, With unidimensional experiments at 360 MHz, quantitative exchange data at’ temperatures near 3ci”C previously obtained for 16 individually assigned arnide protons of BPTI (Wiit,hrich Sr Wagner. 1979: Richarz et 01.. 1979). This already compared favourably with the amount of information on individual exchange rates that can be obtained from experiments other t)han n.m.r. (see e.g. Hvidt Rr Nielsen. 1966; Englander et al.; 1972: Rosa & Richards. 1979). LVith the use of (‘OSY. quantitative exchange rates were obtained for 34 residues, and for four additional residues quantitative data could have been obtained by extension of the duration of the experiment (Table 1). For 14 additional residues, a lower limit for /c,(36”C) was established. Since this limiting rate is only approximately ten times slower than the average exchange rate for exposed amide protons in random coil model peptides (Englander it crl., 1972: and see below), WC are quit’e confident that t,hese 14 prot,ons represent, t,he exchange brhaviour of solvent,-accessible amide protons located near t,hc molecular surface in the solut,ion conformation of the protein. Overall, the COSY peaks of 52 of the total of 53 backbone amide protons had thus been individually assigned and the exchangfa behaviour of these protons was characterized. In the present experiment,s, a quench method was applied: i.e. the sample temperature was lowered before the start of the n.m.r. measurement Bec>ausr of the relatively long recording times needed to obtain a (‘OSY spectrum with workable signal-to-noise ratio. this will quite generally be needed. In a quench experiment. t,he observed resonance intensity. I(f). where t is t,he time during which exchange took place before the reaction was quenched. can be writt,en:
I, is the initial peak intensity. Ic, the exchange rat,e constant of interest,. t,,, the rate constant under tht time used t,o record the spectrum and k,,, t,he exchange conditions that prevail during the recording of the spectrum. Equation (2) yields:
It is readily apparent from protons, with k,,,t,,, -=$ 1, the can be neglected. For the quantitative evaluation of k,
equation (3) that for t,he very slowly exchanging exchange during the recording of the COSY spectrum more rapidly exchanging protons, straightforward is possible when fresh samples are prepared for each
SI’RFACE .~
1
STRI’CTI’KE -
OF
BPTI
IN
3.55
SOLL’TIOX
I
2 0
0
Oo
0 0 P0 G12
A25
1:5 0
2 4 2 5i
0
0
4
5
9
8
7
WI (P.P.rn.1
Fit:. 5. Contour plot ofthe absolute value 500 MHz ‘H COSY spectrum of a @02 M solution of Hl’Tl in ‘H,O, p2H 35, at IWC. Immediately before the n.m.r. experiment, the protein was dissolved in ‘Hz0 at 4°C. The spectrum was recorded in 12 h. The digital resolution is 53 Hz/point. The same spectral region is shown as in Figs 1 and 2. The assignments are indicated for the cross-peaks that could not be observed in the experiments of Figs 2 and 3. The vertical noise bands on the right of the spectrum are tails of the strong aromat,ic signals in the adjacent spectral region.
measurement of I(t), and identical measuring times, t,,,, are used for all recordings of POSY spectra. In this case, the pre-exponent’ial factor in equation (3) is the same for all measurements and the quantitative evaluation of 12, is not affected by the length of the recording time, provided that for each measurement I(t) is X1,/5, which is the practical limit where the signal intensity can still be measured. With rapid quenching of both pH and temperature to the conditions of minimal exchange rate. and provided that the protein is not denatured under t,hese conditions, the COSY experiment can thus be used for amide proton exchange
3%
(:. N'A(:SER,
AXI)
K. Wi'THKI(‘H
st,udies over a wide range of experiment,al caondit.ions. The spectrum of Figure 5 demonstrates that well-resolved COSY spectra can br obtained at conditions that are near t)hose for minimal exchange rates (Englander rot 01.. 1972). The fastest exchange brt’ween protons of the protein and deuterons of the solvent *HZ0 that may be measured with the presently described USC of C’OSY at lO’(’ (Fig. 5) is approximately MO5 min- ’ One arrives at this value with the assumption t’hat for quant,itat)ive st)udies the peak intensity after 12 hours at 1WC’ should br 2 Z&. Following equation (2) of l3nglander r>t (11. (1972). the average exchange rate for solvent-exposed amide protons in model pe@idrs at 10 (’ and p2 H 35 is of the order of 0455 rnin ‘. WC thus know that solvent-exposed amide protons exchange too rapidly to be sevn in (‘OS\- experiments in 2H20. hut the gall between thr limiting observable value and t.hr random c-oil exAanpe rat.r is only of the order of IO. The lower limits of the exvhangv rates at 36 and 68“(‘. respec*tivt~ly. for the protons that exchange too rapidly to 1~ seen at 10°C’. NY&W obtained by ext,rapolation from 10°C to higher temperatures. This means that from a lower limit /c,(lO”C’) > ON!5 min-‘, lower lirnits at lO’(‘, at highrr tjemperatures. k,(BB”(‘) > 0.1 mill- ’ and /z,(SX”C’) > 5 mirC ‘. wt’rt‘ estimated lvith the use of equation (2) of Englandrr rt nl. (1972): i.e. it lvas assumed that the erlthalpy of actjivat,ion for these protons is not lower t,han that of sol~cnt-cxposecl protons in model pept’ides.
(‘omparrd to the classical amide proton exchange measurements. which usv ra.dioaotivc tracers or infrared spectroscopy (Hvidt K: Xi&en. 1966 ; Englander et al.. 1972: Rosa Kr Richards, 1979). n.m.r. has the advantage of heing able to provide quantitative excahange rates for individual amide prot,ons in specGfied locations along the polypeptide chain. As a consequence of the improved spectral resolution in C’OSY spectra as compared to c*onvent’ional unidimensional n.m .I’. much rnorr cxt,ensive set,s of individual exchange rat,es can be measured. In BPTT, a complete mapping of thP amid? proton exchange rates for the ent’ire polypeptide chain was t bus obtained. &An alt,crnativr. novel procedure to map amide proton exchange rates in protein uses neutron diffraction techniques (Kossiakoff. 1982). A map of qualitative exchange data was thus recently obtained for c:rystalline t.rypsin (Kossiakoff, 1982). Since neutron diffraction and 21) n.rr1.r. can provide nearly complete mappings of amide proton exchange rat,es in single crystals and in non-crystalline the combined ust~ of the t’wo methods with the same environments. respectirelg. protein should open an avenue for direct comparison of the molecular dynamics in the crystal and in solut,ion. From the available information (Kossiakoff. 1982). the neutron diffraction technique should be more readily applicable for bigger proteins t,han 21) n.rn.r., but it might not be practical for proteins of any size t,o obtain quantitative exchange rat’es from neutron diffraction experiments. COSY can provide quantitat,ive c,xchangr measurements under a variety of different experirnental conditions. which will probably be of considerable import,anve for invest~igations of mechanistic asprds of prot.on exchange (see below). (‘onsidering
SI.RF.A(‘E
S’1’RI’(‘1‘17RE
OF
R1’1’1
IS
SOI,17’I’IOS
35;
that the use of both techniques for prot,on exchange studies is just being introduced. it, would appear premature to evaluate further their respective potentialities. but already now there appears t,o be a good chance that they could in many wa.vs provide complementary data on details of internal dynamics of proteins tha,t ww hitherto not amenable to experiment’al investigations.
The availability of complet,e sets of individually assigned amide proton exchange rates measured wit’h different conditions of pH and temperature should in the future help to clarify some uncertainties concerning the mechanisms 1)~ which t,he proton exchange reactions in BPTT proceed (Hilton & IVoodward, 1978.1979: LYagner & Wiithrich. 1979; Wtithrich d nl.. 1980n,b: Hilton et nl.. 1981). Here, n-e would like to describe a preliminary observation that might’ have some bearing on our understanding of exchange mechanisms. In Figure 6. the individual amide proton exchange rates for BPTI at 68°C’ and pH 3.5 are presented together with the intrinsic exchange rates. The latter are exchange rat’es for t’he random coil form of t,he polyprptide chain, which manifest the s~~~uen~~e cffert’s on the exchange of solvent-acc,essiI,le protons. Intrinsic rate constants f’or BPTI at 68°C’ and p2H 35 were calculat~ed wit,h the rules of Molda? rt nl. (197%). Figure 6 clearly shows that there is no simple relation between the two sets of rate constants and it can in most in&am-es be excluded that t,he relative values of’ thr exchange rates, k,, of neighbowing residues in the amino acid sequence are determined by seqnrnc~e effthcts. On the other hand. in an overall procws dominated by exchange bctwwn denatured prot’ein and the solvent one would intnit,ivcly expect the relative rates for individual neighboring protons to be
358
(:.
W$GNER
AND
K.
W~THRl(‘H
dominated by the intrinsic exchange rates. On this basis, the observations in Figure 6 might be forwarded as evidence against an exchange mechanism dominated by admixture of denatured protein molecules to the ensemble of BPTI conformers present in aqueous solution at 68°C (Hilton et al., 1981). (d) Crystal
structure
of
RPTI
and
amide
proton
exchange
in
solution
Figure 7 presents the accessible surface areas (Lee & Richards, 1971) for the backbone amide groups in the BPTI crystal structure (Deisenhofer & Steigemann, 1975) and the proton exchange rates in solution at two different temperatures. Overall, the qualitative similarity of the patterns obtained in the three graphs representing the accessible surface areas, k,(36”C) and k,(SS”C) along the sequence is quite striking. If one takes the pat!terns of exchange rates as an empirical manifestation of the three-dimensional protein structure, then the close similarity between the data obtained at 36°C and 68°C would indicate that the exchange at these two temperatures is from closely similar conformations. It would thus appear that Figure 7 provides additional evidence against the hypothesis that amide proton exchange at higher temperature is dominated by exchange from the denatured protein (Hilton et al., 1981). Some intriguing observations result from evaluation of the exchange rates in the light of the hydrogen-bonding network in the crystal structure. The conclusion that the exchange in the p-sheet is faster at both ends than in the central region: which resulted previously from a limited number of individually assigned exchange rates, is confirmed by the present, complet,e dat,a set. The most slowly exchanging protons are those of residues 21 to 24, which form the central strand in a short region of triple-stranded p-sheet (Deisenhofer bt. Steigemann, 1975). Except for Ala16, the exchange rates at 36°C of all other hydrogen-bonded amide protons in the p-sheet tend to be slower than t,hose for the hydrogen-bonded protons in the n-helix. The individual protons in the n-helix have also somewhat different, rates. whereby the exchange is slowest for Cys55 and Met52. Quite possibly. this is an effect of the disulphide bonds formed by Cys51 and (‘~~5.5. Further. it seems worth not,ing that the amide protons of TyrlO, Cysl4, Cys38 and Lys41, which in the crystal structure are hydrogen-bonded with internal water molecules (Fig. 7: Deisenhofer & Steigemann, 1975), do not, have unusual exchange rates. As one might have anticipated from the vanishing or very small (Lys41) static accessible surface areas for these residues in the crystal structure. we observed exchange rates k,(36”C) < 0.1 min-’ (Table 1 : Fig. 7). The exchange studies of amide protons that are located near the protein surface in the single crystal structure should be a useful tool to probe the surface of the solution conformation. One might, in a preliminary evaluation for example, assume that all the amide groups that have a non-vanishing accessible surface area, and are hence in van der Waals’ contact with the solvent (Lee &, Richards, 1971), should exchange rapidly; i.c. with &,(36”(Y) > 0.1 min-‘. k,(36”C) values would accordingly correspond to groups with zero accessible surface 10.1 min-’ area. Figure 7 and Table 1 reveal that there is a single residue that has zero accessible surface area and k,(36”C) > 0.1 min - ’ : i.e. Glu49. If one assumes that
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the protein surface is dynamic in the sense that the atoms undergo fluctuations about an equilibrium position, it is quite conceivable that’ the solvent exposure is increased as compared to the static accessibility in the crystal st,ructure (Lee 6: Richards, 1971). On the other hand, there are ten residues that would in the crystal structure be in van der Waals’ contact wit,h the solvent but, have in solut,ion. Theke are Asp3. Glu7. Gly12, Alal6. Ala25. k,(36”C) I 0.1 mind1 Gly28, Cys30, Thr32, Va134 and Lys41. Here it appears intuitively rather unlikely that dynamic fluctuations of the protein structure could reduce the soIlvent accessibility as compared to the static accessibility in the crystal, and there is an indication that these residues are in different environments in the cryst,al and in solution The solvent-accessible surface area for the crystal structure (Table 1) was computed for an isolated BPTI molecule. Hence, lack of correlation with the amide proton exchange rates in solution could be due either to intermolecular aggregation at the high protein concentrations for which t,he data in Table 1 and Figure 7 were obtained, or to rearrangement, of the protein surface structure between the crystal and t’he solution. Further studies of the apparent, discrepancies between the conformations of BPTI in single crystals and in solution are in progress. These will include comparison of the proton exchange data in Table 1 with corresponding measurements of exchange rates in very dilute solutions of BPTI, to distinguish between influences of intermolecular aggregation and conformational changes in monomeric BPTI. We envisage that the observation of apparent, discarepancies between the structures of the protein surfaces in the c=rystal and in solution by amide proton exchange studies could be a start,ing point for site-specific use of different, more laborious n.m.r. experiments capable of providing direct information on the surface conformation in these locations and/or on struc*tural details of intermolecular aggregation in concentrated protein solutions. We thank the Schweizerische Nationalfonds (project 3.538.7!)) for financial support. the Fnrhenfahriken Bayer A(: for a generous gift of Bl’TI (T rasylol”), I)r P. (‘hothia for a complete listing of accessible surface areas in KI’TI. and ,Mrs E. Huber and Xlrs E:. H. Hunziker for the careful preparation of the manuwript and the illustrations.
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STRV(“l’L’HE
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SOI,I’TIO?1’
36 I
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by
I’. Luzzati