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Faraday Discuss., 1992,93, 35-45
Protein Hydration in Aqueous Solution Kurt Wuthrich,* Gottfried Otting and Edvards Liepinsh Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische HochschuleHonggerberg, CH-8093 Zurich, Switzerland
Proton nuclear magnetic resonance was used to study individual molecules of hydration water bound to the protein basic pancreatic trypsin inhibitor (BPTI) and to the nonapeptide oxytocin in aqueous solution. The experimental observations are nuclear Overhauser effects (NOE) between protons of individual amino acid residues of the protein and those of hydration water. These NOEs were recorded by two-dimensional (2D) and three dimensional (3D) NOE spectroscopy (NOESY) in the laboratory frame, and by the corresponding experiments in the rotating frame (ROESY). The studies show that there are two qualitatively different types of hydration sites. Four water molecules in the interior of the BPTI molecule are in identical locations in the crystal structure and in solution. Their NOEs with the protein protons are characterized by large negative cross-relaxationrates uNOE , which indicates that the residence times of the water molecules in these hydration sites are longer than ca. 10 ns. Additional experiments with extrinsic shift reagents established an upper limit of 20 ms at 4 "C for these residence times. Surface hydration of both the globular protein BPTI and the flexibly disordered polypeptide oxytocin is by water molecules with residence times in the subnanosecond range, as evidenced by small positive uNOE values observed for their NOEs with nearby polypeptide protons. Short residence times prevail for all surface hydration sites, independent of whether or not they are occupied by well ordered, X-ray observable water in the protein single crystals.
Three-dimensional protein structures can be determined either by X-ray diffraction in single crystals' or by nuclear magnetic resonance (NMR) spectroscopy in solution.2 High-resolution crystal structures of globular proteins include typically numerous water molecules in defined hydration sites. The Brookhaven Protein Data Bank3thus includes the locations of over 30 000 water oxygen atoms. The large majority of these water sites are on the molecular surface. In addition, a small number of water molecules may be located in the interior of a protein and represent an integral part of the molecular architecture. Although the NMR method for protein structure determination has been available since 1985: hydration water molecules proved to be evasive to detection in aqueous solution and the observation of individual water molecules in a globular protein was first reported in 1989, when NOE cross-peaks between individual polypeptide protons of the basic pancreatic trypsin inhibitor (BPTI) and protons of four interior water molecules could be identifiedS4These four water molecules are also present in the three available X-ray crystal structures of BPTIY5-'with conserved hydrogen-bonding partners and zero solvent accessibility to water molecules outside the protein molecule. Later on, identification of one, eleven and four interior water molecules, respectively, was reported from NMR observations in aqueous solution of a scorpion toxin from Androctonus australis Hector,' interleukin l p' and reduced human thioredoxin. lo Most recently, individual hydration water molecules in surface sites could be identified." The results obtained made clear why the detection of surface hydration water molecules by NMR 35
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36
Protein Hydration in Aqueous Solution
in solution is particularly difficult, since the surface waters have very short residence times and hence greatly reduced NOE intensities with the protein protons. The present manuscript gives an overview of the information accessible by high-resolution NMR observation of individual hydration water molecules in aqueous protein solutions, using the NMR solution data and X-ray crystal data available for BPTI and the polypeptide hormone oxytocin as illustrations.
General Considerations on the Observation of Individual Molecules of Hydration Water by 'HNMR On grounds of principle one would anticipate that the proton chemical shifts of proteinbound water molecules are influenced by the protein environment in a similar fashion as those of the individual amino acid residues upon incorporation into a globular protein structure.2 On the basis of the resulting unique chemical shifts for different individual hydration waters, direct observation of distinct 'H NMR lines corresponding to these water molecules should in principle be possible and thus provide direct evidence for their presence. The experience gained with all systems studied so far indicates, however, that the proton resonances of protein hydration waters in aqueous solution are usually at the same chemical shift as the bulk water. Experiments with paramagnetic chemical shift reagents (see below) showed that this is due to rapid exchange between hydration water and bulk water, which averages the differences in chemical shift induced by the different chemical environments in the bulk solution and the protein hydration sites. The same applies for nearly all hydroxyl and carboxylate protons of the amino acid side chains of Ser, Thr, Tyr, Asp and Glu in proteins, for which it is well known that they have intrinsically different chemical shifts from that of H20.*Since different rate processes with largely different timescales govern the averaging of the chemical shifts or the nuclear spin relaxation that leads to the appearance of NOEs, specific intermolecular NOEs with individual molecules of hydration water may still be observed in the situation where the resonance lines of the protein OH groups and the hydration waters are merged with that of the bulk water. However, the fact that all water protons and most hydroxyl protons have the same chemical shift then requires that apart from the 'H NMR assignments the solution structure of the protein must be available at high resolution before unambiguous assignments of NOEs between protons of the protein and hydration water molecules can be obtained. In addition, one must rule out that the observed effects arise from chemical exchange of protons, and that the interactions could be with OH groups of the protein rather than with protein-bound water molecules. Different possible mechanisms of incoherent magnetization transfer can be distinguished from differences in qualitative aspects of two-dimensional nuclear Overhauser experiments in the rotating frame (ROESY). A negative sign of a ROESY cross-peak relative to the diagonal peaks shows that the magnetization transfer is by direct crossrelaxation, since both transfer by first-order s in diffusion pathways or by chemical exchange would lead to positive cross-peaks.14 Once a cross-peak between a protein proton and a proton at the bulk water chemical shift has thus been demonstrated to correspond to a direct NOE, distinction between intermolecular NOEs with hydration water and intramolecular NOEs with OH groups can in favourable cases be achieved on the basis of the solution structure of the protein, from which the location of all the side-chain hydroxy and carboxy groups is known. Because sizeable NOEs can be observed only for proton-proton distances shorter than ca. 4.0& an NOE with a hydration water molecule is indicated whenever the interacting protein proton is at a distance >4.0A from the nearest OH group. Along similar lines, a distinction may be made between the two situations that either one hydration water molecule interacts with two nearby groups of polypeptide protons, or that two different water molecules interact with the different groups of protein protons.
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37
9
1 -1 I . : : : : : : : ; : : 1 3 5 7 9 1
1
PH Fig. 1 Logarithmic plots of approximate exchange rate constants, kintr,for solvent-accessible, labile protons of polypeptides in H 2 0 solution at 25 "C 0s. pH. Broken lines represent lower limits for kintrin situations where pK, data were available only for base catalysis. The individual curves are identified with the proton types and, where applicable, the residues types (Im stands for imidazole ring NH, Gua for guanidinium NH, bb for backbone). Reprinted by permission of John Wiley & Sons, Inc., from K. Wiithrich, NMR of Proteins and Nucleic Acids, John Wiley, New York, 1986. @ Copyright 1986 John Wiley & Sons Inc.
An unambiguous identification of intramolecular NOEs with side-chain OH groups is achieved when the chemical exchange of the hydroxyl protons can be made sufficiently slow to enable the observation of separate signals away from the water line. The slowest exchange rates are expected near the pH value where the acid-catalysed and basecatalysed exchange have approximately the same rates. Fig. 1 shows plots of the exchange rates us. pH for different types of labile protons occurring in polypeptide chains. The curves were computed from the pK, values of the reactants assuming diffusion-limited confirmed that the rate constant^.'^ Direct experimental exchange-rate rnea~urements'~ hydroxy protons of the seryl and threonyl residues in BPTI exchange most slowly near pH 6.0, and the tyrosyl hydroxy protons were found to exchange most slowly near pH 5.0. At these pH values and a temperature of 4"C, all labile hydroxy protons of the tyrosyl, seryl and threonyl residues of BPTI give rise to individual resonances that are well separated from the water signal, and the NOEs with these protons can be observed as well resolved cross-peaks. However, even under these most favourable conditions the NOEs with the hydroxy protons are usually also observed at the water chemical shift due to magnetization transfer by chemical exchange.
NMR Experiments and Experimental Conditions used for the Observation of Hydration Water Molecules In experiments used for the observation of intermolecular NOEs between water protons and polypeptide protons, no solvent suppression by preirradiation of the dominant water signal can be e m p l ~ y e d .Instead, ~ the water magnetization must be eliminated after the mixing period during which the NOE transfer of magnetization from the water protons to the protein protons has taken place. For observation of surface hydration water molecules the NOEs must be recorded with short mixing times and at low temperature (see below). The most suitable 2D and 3D NOESY and ROESY experiments
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Protein Hydration in Aqueous Solution
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38
co
9 zz III
I
Y
3
t
I
I
10
I
9,
I
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4ppm)
1
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Fig. 2 Cross-sections taken at the w1 chemical shift of the water resonance through two-dimensional nuclear Overhauser enhancement (NOE) spectra recorded in the laboratory frame (NOESY) ( a ) and the rotating frame (ROESY) (b). The spectra were recorded at 600 MHz on a Bruker AM600 spectrometer, using a 20 mmol dm-3 solution of BPTI in 90 % H20-10%D20,pH 3.5,4 "C, with mixing times of 50 ms. Both experiments were conducted as 'soft-NOESY' and 'soft-ROESY' experirnents,l8 respectively, with a single semi-selective inversion pulse centered at the frequency of the water resonance in the middle of the evolution period." In these experiments most of the diagonal peaks are eliminated, which results in a reduction of tl noise and the ensuing baseline artifacts. The water resonance was suppressed with spin-lock pulses as described in ref. 16. The NOE cross-peaks with the water resonance are identified above the NOESY cross-section, using the proton type, the one-letteramino acid symbol and the sequence number in the polypeptide chain
for measurements with short mixing times use spin-lock purge pulses to suppress the water signal. These NMR technical details have been described in detail else~here.'~*'~ In 2D NOESY and ROESY experiments the NOEs with the protons at the bulk-water chemical shift are all located on the 1D cross-section through the water line, which is then quite crowded with lines. Improved resolution can be obtained with homonuclear or heteronuclear 3D NMR experiments. Homonuclear 'H 3D NOESY-TOCSY and 3D ROESY-TOCSY are particularly powerful experiments to assign overlapping crosspeaks with the water line, but they have relatively poor sensitivity when compared to heteronuclear 3D NMR experiments. Heteronuclear correlated 3D NOESY and 3D ROESY experiments have a very good sensitivity with l3C-or "N-enriched proteins, but with these experiments information is obtained only about NOEs with those protein protons that are bound to I3Cor "N, respe~tively?*'~*'~ The results on BPTI and oxytocin described in this article resulted exclusively from homonuclear 'PI NMR experiments. Chemical exchange cross-peaks between the water signal and the resonances of labile polypeptide protons can be minimized by measuring at low temperature and suitably chosen pH values (Fig. 1). With BPTI, the amide proton exchange rates are slowest around pH 3.5, whereas the exchange of the lysyl side chain NH: groups is slower at lower pH values and, as mentioned in the preceding section, the hydroxy protons of the side chains of tyrosine, threonine and serine are best observed at pH values around 5.5. Fig. 2 shows cross-sections parallel to the @,-axis taken at the o1frequency of the water resonance in 2D NOESY and ROESY spectra of BPTI. The spectra were recorded
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39
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K . Wuthrich, G. Otting and E. Liepinsh
I
I
4
3
1
I
2
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%(PPm)
Fig. 3 2D cross-sections taken at the o1frequency of the water signal through a homonuclear 3D 'H NOESY-TOCSY spectrum (A) and a 3D 'H ROESY-TOCSY spectrum (B). Same sample and temperature as in Fig. 2, mixing times 50 ms (NOESY), 25 ms (ROESY) and 27 ms (TOCSY), 500 MHz 'H frequency. In A, negative countour levels are plotted with dashed lines. In B, only negative levels were plotted. The peaks are identified with the assignment of the proton interacting with the water resonance. Reproduced with permission from G. Otting, E. Liepinsh, B. T. Farmer I1 and K. Wuthrich, J. Biomol. NMR, 1991, 1, 209
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40
Protein Hydration in Aqueous Solution
at 4 "C and pH 3.5. The chemical shift of the water is 5.01 ppm, which does not coincide with any of the proton resonances of BPTI. Positive peaks in the ROESY cross-section [Fig. 2(b) J are due to chemical exchange of the hydroxy protons of tyrosyl residues near lOppm, and to exchange of the side-chain NH; protons of Lys-41 and Lys-46 at 7.2 and 7.6 ppm, respectively. The NOESY cross-section of Fig. 2( a ) shows positive crosspeaks for chemical exchange as well as for NOEs with long-lived interior water molecules and with the hydroxy protons. Negative NOESY cross-peaks are observed for a few methyl resonances near 0.8 ppm and the sCH3 group of Met-52 at 2.2 ppm, which are in contact with surface hydration water. It is readily seen from Fig. 2 that the negative NOESY cross-peaks are relatively weak and could easily be dominated by overlapping positive cross-peaks. Therefore, short mixing times must be used for their observation to avoid cancellation by spin diffusion of positive magnetization from the cross-peaks with the hydroxy protons and the interior water molecules to the other resonances of the protein. The chances to observe the weak negative NOESY cross-peaks with surface hydration water are significantlyenhanced by the improved spectral resolution in three-dimensional NMR spectra. Fig. 3 shows the spectral region ( 0 2= 0.5-4.7 ppm, to3= 4.7 pmm) of 2D cross-sections taken through a 'H 3D NOESY-TOCSY spectrum and a 'H 3 D ROESYTOCSY spectrum of BPTI, respectively, at the o1frequency of the water resonance. The peaks on the diagonal come from the transfer of magnetization from the water line to the protein resonances during the NOESY or ROESY mixing, respectively. The diagonals of Fig. 3 A and B thus correspond to the cross-sections shown in Fig. 2 ( a ) and (b). Off-diagonal peaks arise because the magnetization precessing at the 0, frequency after the NOESY or ROESY mixing period is further transferred to scalar coupled protons during the TOCSY mixing period. These TOCSY-relayed peaks are then detected at the o3frequency of the scalar coupled protons. Therefore, the well separated off-diagonal peaks provide a convenient way to determine the sign and the assignment of the corresponding diagonal peaks which represents the direct NOEs with the water line.
Measurements of the Lifetimes of Hydration Water Molecules with Respect to Exchange with the Bulk Water A lower limit for the exchange rates of the four interior water molecules in BPTI was obtained from experiments with the paramagnetic shift reagent CoCl, .20 Water molecules bound to Co2+experience large chemical shifts and some broadening of the 'H NMR lines due to the interactions with the unpaired electrons. The exchange of water molecules in and out of the hydration sites of Co2+ is very fast, so that these paramagnetic effects are averaged over the bulk water.21 A 30 mmol dm-3 concentration of Co2+thus causes a 'H shift for the bulk water of ca. 0.25 ppm.20Although the Co2+ ions have no access to the interior water molecules in BPTI, the NOESY cross-peaks with the interior waters were observed at the bulk water chemical shift also after the addition of Co2+, indicating rapid exchange between interior hydration sites and the bulk water. For the exchange rate constant a lower limit of km>50s-' was thus established.20 An upper limit of k , < lo's-' for the interior water molecules is obtained from the fact that the cross-relaxation rate with nearby polypeptide protons, cNoE, is negative values result in positive NOESY cross peaks and vice versa). (note that negative cNoE In contrast, the small positive aNoE values observed between the water resonance and polypeptide protons on the protein surface indicate residence times of the surface hydration waters in the subnanosecond time range. These exchange rate limits were derived from explicit models describing the motions of the vector connecting a water proton with a proton of the protein. For such model considerations it is of pivotal
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K. Wuthrich, G. Otting and E. Liepinsh
41
importance that the cross-relaxation rates in the laboratory frame, uNoE,or in the differ in their functional dependence on the spectral densities, rotating frame, uRoE, J ( w),2.12.22 aNOE
- 6J(2w0)- J ( 0 )
#OE
- 3J( w,)
+ 2J(O)
(1)
(2)
where wo is the Larmor frequency of the protons. Eqn. (1) and (2) show that uRoE is always positive because the spectral densities J ( w ) have finite positive values at all frequencies.22 In contrast, the sign of aNoE depends on the explicit functional forms of J(20,) and J(O), which in turn are related to the rate processes that govern the modulation of the dipole-dipole coupling defined by the length and orientation of the interproton vector. The sign and value of the ratio uNoE : uRoE thus becomes the quantity of prime interest. Analyses of rigid-sphere models, where the surface hydration water is assumed to be part of the protein molecule, and 'wobbling in a cone' where the hydration water molecules are assumed to be flexibly bound to particular hydration sites of the protein, showed that the experimental observation of positive o N O E . aROE values could be rationalized only with the assumption that translational diffusion of the protein and the water is a dominant additional factor in the modulation values are then predicted for relative of the dipole-dipole couplings. Positive aNoE diffusion coefficients, D, greater than 3 x cm2s-' 25 (note that the self-diffusion cm2s-' 2 6 ) . The diffusion coefficients coefficient of pure water at 4 "C is ca. 12 x can be translated into residence times of the hydration water molecules on the protein surface using the Einstein-Smoluchowski relation r=x2/D (3) of 4.0A as the criterion for complete water Defining an average displacement, is positive for lifetimes shorter proton exchange in and out of a hydration site, uNoE than ca. 500ps. This value is much shorter than the lifetime of a proton in a water molecule with respect to exchange by h y d r ~ l y s i s We . ~ ~therefore conclude that positive aNOE values (i.e., negative NOESY cross peaks) manifest rapid exchange of complete water molecules between the bulk solvent and the protein hydration sites.
0,
Survey of the NMR Data on Surface Hydration of the Polypeptide Hormone Oxytocin and the Globular Protein BPTI Oxytocin has a flexible conformation in aqueous solution and contains no interior water molecules. Because of its small size even its one-dimensional 'H NMR spectrum is well resolved, so that the NOESof the individual polypeptide protons with the water resonance are well separated in 2D NOESY and 2D ROESY experiments." In aqueous solution at temperatures below 6°C the overall rotational tumbling of oxytocin is in the slowmotional regime,22with a rotational correlation time longer than 2 ns. Correspondingly, all NOESY cross-peaks between different polypeptide protons are positive. In contrast, negative NOE cross-peaks between the water line and the peptide signals were observed throughout in a NOESY spectrum recorded at 6"C, showing that the residence times of the hydration water molecules are shorter than 500 ps. The fact that all 'H resonances of oxytocin have negative NOESY cross-peaks with the water line (apart from some positive cross-peaks due to chemical exchange) shows that there are no hydration sites which would be occupied by much more stably bound waters than others. The aforementioned conclusions were confirmed by experiments in the temperature range 0--25 "C, using oxytocin in a mixed solvent of 60 % H20-40 % [2H]acetone.14 At all temperatures, positive NOESY cross-peaks were observed between different peptide protons. In contrast, the sign of the NOE cross-peaks between the water protons
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Protein Hydration in Aqueous Solution
and the protons of the polypeptide is negative above 0 "C and positive at -25 "C. At some intermediate temperature the NOESY cross-peaks vanish, while the intensity of the ROESY cross-peaks increases continuously with decreasing temperature. For different polypeptide protons the sign inversion of aNoE occurs at different temperatures, showing that there are variations of the hydration water residence times in different sites on the protein surface. From these low-temperature experiments there was again no evidence for hydration water that would be bound to oxytocin with a lifetime exceeding 500 ps at temperatures above 0 "C. Based on the experience gained with oxytocin, one might expect that most or all polypeptide protons near the surface of globular proteins should show negative NOESY cross-peaks with the water, So far, in BPTI only a limited number of these expected NOEs have been observed (Fig. 3). We attribute the apparent absence of many of the expected NOESY cross-peaks to the low sensitivity of the experiments used. Most of the negative cross-peaks seen in Fig. 2 and 3 are with intense resonances of BPTI, such as those of methyl groups. This sensitivity criterion applies, however, only to weak NOEs from short-lived hydration waters. Long-lived surface hydration waters would produce much stronger, positive NOESY cross-peaks, comparable to those seen for the internal waters. It is therefore very unlikely that NOEs with surface hydration water molecules bound with residence times >500 ps would have escaped detection by both NOESY and ROESY.
Discussion: Comparison of the X-Ray Diffraction Data on Individual Hydration Water Molecules in BPTI Crystals and the Corresponding NMR Data obtained in Aqueous Solution NMR experiments in solution and X-ray diffraction experiments with protein single crystals are sensitive to different aspects of protein hydration. The sign and intensity of the protein-water NOEs observed by NMR reflect primarily the residence times of the water molecules near the protein protons. In contrast, X-ray diffraction probes the total fraction of time that a water molecule spends at a particular point in space, but is largely insensitive to the residence time at that site on any particular visit.28 On this basis it is of special interest to investigate how the presence or absence of ordered, X-ray observable hydration water in the single crystals can be correlated with the residence times of water molecules in corresponding hydration sites as observed by NMR in solution. BPTI has the same molecular architecture in solution and in crystals, which includes the four internal water molecule^.^-^^^^ In Plate 1the crystal-structure atomic coordinates 5PT13*6were used to generate molecular models, visualizing selected surface properties of the protein in the crystals, and the X-ray and NMR observations on surface hydration. The 'back view' shown is representative of the complete protein surface (the 'front view' was previously presented in ref. 11). With the exception of the protein-protein contact sites in the crystal lattice [yellow in Plate l(a)], the entire protein surface must be covered with hydration water molecules. Only part of the hydration water has so far been observed by either of the two methods considered here. In the BPTI crystal structure, ca. 40 % of the protein surface is involved in protein-protein contacts, 25 % is covered with X-ray-observable hydration waters attributed to the central protein molecule, and 15 % make contacts with X-ray observable water molecules attributed as hydration waters to neighbouring protein molecules [Plate l(b)]. The remaining 20 % of the protein surface must be in contact with water molecules that are not sufficiently well ordered to be seen by X-ray diffraction experiments [comparison of Plate l(a), where white indicates water-accessible hydrogen atoms in the crystal lattice, with Plate l ( b ) , which shows clearly that numerous accessible sites are not covered with X-ray-observable waters). It is further worth noting that more than 40 % of the X-ray-
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(c)
Plate 1. Stereo views of a space-filling CPK representation of the crystal structure 5P"I of BPTI,6 using the following colour codes to visualize salient features of the structure in crystals and in solution. ( a ) Protein surface. Hydrogen, carbon and sulfur atoms are coloured grey, nitrogens blue and oxygens red; hydrogen atoms with more than 20% solvent-accessible surface area in the crystal structure are white and hydrogen atoms within a distance of S 3 . 0 8 , from neighbouring protein molecules in the crystal lattice are yellow. ( b ) Hydration observed in crystals. All polypeptide atoms are grey, the hydration water molecules attributed to this protein molecule in the crystal are green, additional water molecules attributed to hydration sites on neighbouring protein molecules yet located within 3.0 8, are blue-green. (c) NMR observations in aqueous solution. Hydrogen atoms for which 'H-'H NOE cross-peaks with the water resonance were observed are either brown (positive NOESY cross-peaks) or yellow (negative NOESY cross-peaks); polypeptide atom groups with proton chemical shifts at the water resonance, i e . the side-chain hydroxy protons, the N-terminal amino protons and the carboxyl oxygens are coloured magenta; interior hydration waters and the surface-bound water molecule W129, which was observed identically in all three crystal structures5-' (see text) are green; the hydrogen bonding partner of W129, i.e. the amide proton of Ile-19, is red
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observable water molecules are in contact with two protein molecules in the crystal lattice and that all observed waters are in contact with at least one protein molecule, but that most of the observed surface hydration water molecules are also accessible for contact with X-ray unobservable water in the crystal. In aqueous solution all polypeptide hydrogen atoms giving rise to negative cNoE values with the water protons [brown in Plate 1(c)] are located either near the N-terminus, a carboxy group, a hydroxy proton [magenta in Plate l(c)], or near one of the four interior water molecules [green in Plate 1(c)]. From the ensemble of all the experimental observations described in the preceding sections, we arrive at two important points to be made on surface hydration in solution: (i) nearly all positive NOESY cross-peaks between the water resonance and resonances of polypeptide protons on the protein surface are due to the hydroxy protons of serine, threonine and tyrosine, rather than to stably bound water molecules; (ii) the surface hydration sites containing well ordered, X-ray-observable water molecules in the crystal structure have similar residence times for hydration waters in solution as other surface areas for which the hydration water is disordered in the crystals and not observable by X-ray diffraction. Point (i) is supported by the aforementioned experiments in the pH range 5.0-6.5, which showed that almost all of the polypeptide protons near the protein surface that have positive NOESY cross-peaks with the water resonance at more acidic pH give rise to strong NOEs with the hydroxy proton resonances of Ser, Thr and Tyr. Point (ii) results from two independent observations. First, out of a total of ca. 60 X-ray-observable surface hydration waters there are only six water molecules that have conserved hydrogen-bonding partners in all three single-crystal structures of BPTI.5-7 Of those, the hydration sites W143 and W129 [Plate l(c)] could so far be characterized by the NMR data. In the crystal structures, W143 is in hydrogen-bonding distance to the amide proton of Ala-25, which has vanishing cross-peak intensity with the water line in the NOESY experiment and a weak NOE cross-peak in the ROESY spectrum. W129 is within hydrogen-bonding distance of the amide proton of Ile-19, which shows a negative NOESY cross-peak with the water. From these data upper limits for the residence times of
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Faraday Discuss., 1992,93, 35-45
Protein Hydration in Aqueous Solution Kurt Wuthrich,* Gottfried Otting and Edvards Liepinsh Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische HochschuleHonggerberg, CH-8093 Zurich, Switzerland
Proton nuclear magnetic resonance was used to study individual molecules of hydration water bound to the protein basic pancreatic trypsin inhibitor (BPTI) and to the nonapeptide oxytocin in aqueous solution. The experimental observations are nuclear Overhauser effects (NOE) between protons of individual amino acid residues of the protein and those of hydration water. These NOEs were recorded by two-dimensional (2D) and three dimensional (3D) NOE spectroscopy (NOESY) in the laboratory frame, and by the corresponding experiments in the rotating frame (ROESY). The studies show that there are two qualitatively different types of hydration sites. Four water molecules in the interior of the BPTI molecule are in identical locations in the crystal structure and in solution. Their NOEs with the protein protons are characterized by large negative cross-relaxationrates uNOE , which indicates that the residence times of the water molecules in these hydration sites are longer than ca. 10 ns. Additional experiments with extrinsic shift reagents established an upper limit of 20 ms at 4 "C for these residence times. Surface hydration of both the globular protein BPTI and the flexibly disordered polypeptide oxytocin is by water molecules with residence times in the subnanosecond range, as evidenced by small positive uNOE values observed for their NOEs with nearby polypeptide protons. Short residence times prevail for all surface hydration sites, independent of whether or not they are occupied by well ordered, X-ray observable water in the protein single crystals.
Three-dimensional protein structures can be determined either by X-ray diffraction in single crystals' or by nuclear magnetic resonance (NMR) spectroscopy in solution.2 High-resolution crystal structures of globular proteins include typically numerous water molecules in defined hydration sites. The Brookhaven Protein Data Bank3thus includes the locations of over 30 000 water oxygen atoms. The large majority of these water sites are on the molecular surface. In addition, a small number of water molecules may be located in the interior of a protein and represent an integral part of the molecular architecture. Although the NMR method for protein structure determination has been available since 1985: hydration water molecules proved to be evasive to detection in aqueous solution and the observation of individual water molecules in a globular protein was first reported in 1989, when NOE cross-peaks between individual polypeptide protons of the basic pancreatic trypsin inhibitor (BPTI) and protons of four interior water molecules could be identifiedS4These four water molecules are also present in the three available X-ray crystal structures of BPTIY5-'with conserved hydrogen-bonding partners and zero solvent accessibility to water molecules outside the protein molecule. Later on, identification of one, eleven and four interior water molecules, respectively, was reported from NMR observations in aqueous solution of a scorpion toxin from Androctonus australis Hector,' interleukin l p' and reduced human thioredoxin. lo Most recently, individual hydration water molecules in surface sites could be identified." The results obtained made clear why the detection of surface hydration water molecules by NMR 35
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Protein Hydration in Aqueous Solution
in solution is particularly difficult, since the surface waters have very short residence times and hence greatly reduced NOE intensities with the protein protons. The present manuscript gives an overview of the information accessible by high-resolution NMR observation of individual hydration water molecules in aqueous protein solutions, using the NMR solution data and X-ray crystal data available for BPTI and the polypeptide hormone oxytocin as illustrations.
General Considerations on the Observation of Individual Molecules of Hydration Water by 'HNMR On grounds of principle one would anticipate that the proton chemical shifts of proteinbound water molecules are influenced by the protein environment in a similar fashion as those of the individual amino acid residues upon incorporation into a globular protein structure.2 On the basis of the resulting unique chemical shifts for different individual hydration waters, direct observation of distinct 'H NMR lines corresponding to these water molecules should in principle be possible and thus provide direct evidence for their presence. The experience gained with all systems studied so far indicates, however, that the proton resonances of protein hydration waters in aqueous solution are usually at the same chemical shift as the bulk water. Experiments with paramagnetic chemical shift reagents (see below) showed that this is due to rapid exchange between hydration water and bulk water, which averages the differences in chemical shift induced by the different chemical environments in the bulk solution and the protein hydration sites. The same applies for nearly all hydroxyl and carboxylate protons of the amino acid side chains of Ser, Thr, Tyr, Asp and Glu in proteins, for which it is well known that they have intrinsically different chemical shifts from that of H20.*Since different rate processes with largely different timescales govern the averaging of the chemical shifts or the nuclear spin relaxation that leads to the appearance of NOEs, specific intermolecular NOEs with individual molecules of hydration water may still be observed in the situation where the resonance lines of the protein OH groups and the hydration waters are merged with that of the bulk water. However, the fact that all water protons and most hydroxyl protons have the same chemical shift then requires that apart from the 'H NMR assignments the solution structure of the protein must be available at high resolution before unambiguous assignments of NOEs between protons of the protein and hydration water molecules can be obtained. In addition, one must rule out that the observed effects arise from chemical exchange of protons, and that the interactions could be with OH groups of the protein rather than with protein-bound water molecules. Different possible mechanisms of incoherent magnetization transfer can be distinguished from differences in qualitative aspects of two-dimensional nuclear Overhauser experiments in the rotating frame (ROESY). A negative sign of a ROESY cross-peak relative to the diagonal peaks shows that the magnetization transfer is by direct crossrelaxation, since both transfer by first-order s in diffusion pathways or by chemical exchange would lead to positive cross-peaks.14 Once a cross-peak between a protein proton and a proton at the bulk water chemical shift has thus been demonstrated to correspond to a direct NOE, distinction between intermolecular NOEs with hydration water and intramolecular NOEs with OH groups can in favourable cases be achieved on the basis of the solution structure of the protein, from which the location of all the side-chain hydroxy and carboxy groups is known. Because sizeable NOEs can be observed only for proton-proton distances shorter than ca. 4.0& an NOE with a hydration water molecule is indicated whenever the interacting protein proton is at a distance >4.0A from the nearest OH group. Along similar lines, a distinction may be made between the two situations that either one hydration water molecule interacts with two nearby groups of polypeptide protons, or that two different water molecules interact with the different groups of protein protons.
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37
9
1 -1 I . : : : : : : : ; : : 1 3 5 7 9 1
1
PH Fig. 1 Logarithmic plots of approximate exchange rate constants, kintr,for solvent-accessible, labile protons of polypeptides in H 2 0 solution at 25 "C 0s. pH. Broken lines represent lower limits for kintrin situations where pK, data were available only for base catalysis. The individual curves are identified with the proton types and, where applicable, the residues types (Im stands for imidazole ring NH, Gua for guanidinium NH, bb for backbone). Reprinted by permission of John Wiley & Sons, Inc., from K. Wiithrich, NMR of Proteins and Nucleic Acids, John Wiley, New York, 1986. @ Copyright 1986 John Wiley & Sons Inc.
An unambiguous identification of intramolecular NOEs with side-chain OH groups is achieved when the chemical exchange of the hydroxyl protons can be made sufficiently slow to enable the observation of separate signals away from the water line. The slowest exchange rates are expected near the pH value where the acid-catalysed and basecatalysed exchange have approximately the same rates. Fig. 1 shows plots of the exchange rates us. pH for different types of labile protons occurring in polypeptide chains. The curves were computed from the pK, values of the reactants assuming diffusion-limited confirmed that the rate constant^.'^ Direct experimental exchange-rate rnea~urements'~ hydroxy protons of the seryl and threonyl residues in BPTI exchange most slowly near pH 6.0, and the tyrosyl hydroxy protons were found to exchange most slowly near pH 5.0. At these pH values and a temperature of 4"C, all labile hydroxy protons of the tyrosyl, seryl and threonyl residues of BPTI give rise to individual resonances that are well separated from the water signal, and the NOEs with these protons can be observed as well resolved cross-peaks. However, even under these most favourable conditions the NOEs with the hydroxy protons are usually also observed at the water chemical shift due to magnetization transfer by chemical exchange.
NMR Experiments and Experimental Conditions used for the Observation of Hydration Water Molecules In experiments used for the observation of intermolecular NOEs between water protons and polypeptide protons, no solvent suppression by preirradiation of the dominant water signal can be e m p l ~ y e d .Instead, ~ the water magnetization must be eliminated after the mixing period during which the NOE transfer of magnetization from the water protons to the protein protons has taken place. For observation of surface hydration water molecules the NOEs must be recorded with short mixing times and at low temperature (see below). The most suitable 2D and 3D NOESY and ROESY experiments
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Protein Hydration in Aqueous Solution
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38
co
9 zz III
I
Y
3
t
I
I
10
I
9,
I
I
8
7
4ppm)
1
I
i
1
4
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Fig. 2 Cross-sections taken at the w1 chemical shift of the water resonance through two-dimensional nuclear Overhauser enhancement (NOE) spectra recorded in the laboratory frame (NOESY) ( a ) and the rotating frame (ROESY) (b). The spectra were recorded at 600 MHz on a Bruker AM600 spectrometer, using a 20 mmol dm-3 solution of BPTI in 90 % H20-10%D20,pH 3.5,4 "C, with mixing times of 50 ms. Both experiments were conducted as 'soft-NOESY' and 'soft-ROESY' experirnents,l8 respectively, with a single semi-selective inversion pulse centered at the frequency of the water resonance in the middle of the evolution period." In these experiments most of the diagonal peaks are eliminated, which results in a reduction of tl noise and the ensuing baseline artifacts. The water resonance was suppressed with spin-lock pulses as described in ref. 16. The NOE cross-peaks with the water resonance are identified above the NOESY cross-section, using the proton type, the one-letteramino acid symbol and the sequence number in the polypeptide chain
for measurements with short mixing times use spin-lock purge pulses to suppress the water signal. These NMR technical details have been described in detail else~here.'~*'~ In 2D NOESY and ROESY experiments the NOEs with the protons at the bulk-water chemical shift are all located on the 1D cross-section through the water line, which is then quite crowded with lines. Improved resolution can be obtained with homonuclear or heteronuclear 3D NMR experiments. Homonuclear 'H 3D NOESY-TOCSY and 3D ROESY-TOCSY are particularly powerful experiments to assign overlapping crosspeaks with the water line, but they have relatively poor sensitivity when compared to heteronuclear 3D NMR experiments. Heteronuclear correlated 3D NOESY and 3D ROESY experiments have a very good sensitivity with l3C-or "N-enriched proteins, but with these experiments information is obtained only about NOEs with those protein protons that are bound to I3Cor "N, respe~tively?*'~*'~ The results on BPTI and oxytocin described in this article resulted exclusively from homonuclear 'PI NMR experiments. Chemical exchange cross-peaks between the water signal and the resonances of labile polypeptide protons can be minimized by measuring at low temperature and suitably chosen pH values (Fig. 1). With BPTI, the amide proton exchange rates are slowest around pH 3.5, whereas the exchange of the lysyl side chain NH: groups is slower at lower pH values and, as mentioned in the preceding section, the hydroxy protons of the side chains of tyrosine, threonine and serine are best observed at pH values around 5.5. Fig. 2 shows cross-sections parallel to the @,-axis taken at the o1frequency of the water resonance in 2D NOESY and ROESY spectra of BPTI. The spectra were recorded
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39
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K . Wuthrich, G. Otting and E. Liepinsh
I
I
4
3
1
I
2
1
0
%(PPm)
Fig. 3 2D cross-sections taken at the o1frequency of the water signal through a homonuclear 3D 'H NOESY-TOCSY spectrum (A) and a 3D 'H ROESY-TOCSY spectrum (B). Same sample and temperature as in Fig. 2, mixing times 50 ms (NOESY), 25 ms (ROESY) and 27 ms (TOCSY), 500 MHz 'H frequency. In A, negative countour levels are plotted with dashed lines. In B, only negative levels were plotted. The peaks are identified with the assignment of the proton interacting with the water resonance. Reproduced with permission from G. Otting, E. Liepinsh, B. T. Farmer I1 and K. Wuthrich, J. Biomol. NMR, 1991, 1, 209
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40
Protein Hydration in Aqueous Solution
at 4 "C and pH 3.5. The chemical shift of the water is 5.01 ppm, which does not coincide with any of the proton resonances of BPTI. Positive peaks in the ROESY cross-section [Fig. 2(b) J are due to chemical exchange of the hydroxy protons of tyrosyl residues near lOppm, and to exchange of the side-chain NH; protons of Lys-41 and Lys-46 at 7.2 and 7.6 ppm, respectively. The NOESY cross-section of Fig. 2( a ) shows positive crosspeaks for chemical exchange as well as for NOEs with long-lived interior water molecules and with the hydroxy protons. Negative NOESY cross-peaks are observed for a few methyl resonances near 0.8 ppm and the sCH3 group of Met-52 at 2.2 ppm, which are in contact with surface hydration water. It is readily seen from Fig. 2 that the negative NOESY cross-peaks are relatively weak and could easily be dominated by overlapping positive cross-peaks. Therefore, short mixing times must be used for their observation to avoid cancellation by spin diffusion of positive magnetization from the cross-peaks with the hydroxy protons and the interior water molecules to the other resonances of the protein. The chances to observe the weak negative NOESY cross-peaks with surface hydration water are significantlyenhanced by the improved spectral resolution in three-dimensional NMR spectra. Fig. 3 shows the spectral region ( 0 2= 0.5-4.7 ppm, to3= 4.7 pmm) of 2D cross-sections taken through a 'H 3D NOESY-TOCSY spectrum and a 'H 3 D ROESYTOCSY spectrum of BPTI, respectively, at the o1frequency of the water resonance. The peaks on the diagonal come from the transfer of magnetization from the water line to the protein resonances during the NOESY or ROESY mixing, respectively. The diagonals of Fig. 3 A and B thus correspond to the cross-sections shown in Fig. 2 ( a ) and (b). Off-diagonal peaks arise because the magnetization precessing at the 0, frequency after the NOESY or ROESY mixing period is further transferred to scalar coupled protons during the TOCSY mixing period. These TOCSY-relayed peaks are then detected at the o3frequency of the scalar coupled protons. Therefore, the well separated off-diagonal peaks provide a convenient way to determine the sign and the assignment of the corresponding diagonal peaks which represents the direct NOEs with the water line.
Measurements of the Lifetimes of Hydration Water Molecules with Respect to Exchange with the Bulk Water A lower limit for the exchange rates of the four interior water molecules in BPTI was obtained from experiments with the paramagnetic shift reagent CoCl, .20 Water molecules bound to Co2+experience large chemical shifts and some broadening of the 'H NMR lines due to the interactions with the unpaired electrons. The exchange of water molecules in and out of the hydration sites of Co2+ is very fast, so that these paramagnetic effects are averaged over the bulk water.21 A 30 mmol dm-3 concentration of Co2+thus causes a 'H shift for the bulk water of ca. 0.25 ppm.20Although the Co2+ ions have no access to the interior water molecules in BPTI, the NOESY cross-peaks with the interior waters were observed at the bulk water chemical shift also after the addition of Co2+, indicating rapid exchange between interior hydration sites and the bulk water. For the exchange rate constant a lower limit of km>50s-' was thus established.20 An upper limit of k , < lo's-' for the interior water molecules is obtained from the fact that the cross-relaxation rate with nearby polypeptide protons, cNoE, is negative values result in positive NOESY cross peaks and vice versa). (note that negative cNoE In contrast, the small positive aNoE values observed between the water resonance and polypeptide protons on the protein surface indicate residence times of the surface hydration waters in the subnanosecond time range. These exchange rate limits were derived from explicit models describing the motions of the vector connecting a water proton with a proton of the protein. For such model considerations it is of pivotal
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importance that the cross-relaxation rates in the laboratory frame, uNoE,or in the differ in their functional dependence on the spectral densities, rotating frame, uRoE, J ( w),2.12.22 aNOE
- 6J(2w0)- J ( 0 )
#OE
- 3J( w,)
+ 2J(O)
(1)
(2)
where wo is the Larmor frequency of the protons. Eqn. (1) and (2) show that uRoE is always positive because the spectral densities J ( w ) have finite positive values at all frequencies.22 In contrast, the sign of aNoE depends on the explicit functional forms of J(20,) and J(O), which in turn are related to the rate processes that govern the modulation of the dipole-dipole coupling defined by the length and orientation of the interproton vector. The sign and value of the ratio uNoE : uRoE thus becomes the quantity of prime interest. Analyses of rigid-sphere models, where the surface hydration water is assumed to be part of the protein molecule, and 'wobbling in a cone' where the hydration water molecules are assumed to be flexibly bound to particular hydration sites of the protein, showed that the experimental observation of positive o N O E . aROE values could be rationalized only with the assumption that translational diffusion of the protein and the water is a dominant additional factor in the modulation values are then predicted for relative of the dipole-dipole couplings. Positive aNoE diffusion coefficients, D, greater than 3 x cm2s-' 25 (note that the self-diffusion cm2s-' 2 6 ) . The diffusion coefficients coefficient of pure water at 4 "C is ca. 12 x can be translated into residence times of the hydration water molecules on the protein surface using the Einstein-Smoluchowski relation r=x2/D (3) of 4.0A as the criterion for complete water Defining an average displacement, is positive for lifetimes shorter proton exchange in and out of a hydration site, uNoE than ca. 500ps. This value is much shorter than the lifetime of a proton in a water molecule with respect to exchange by h y d r ~ l y s i s We . ~ ~therefore conclude that positive aNOE values (i.e., negative NOESY cross peaks) manifest rapid exchange of complete water molecules between the bulk solvent and the protein hydration sites.
0,
Survey of the NMR Data on Surface Hydration of the Polypeptide Hormone Oxytocin and the Globular Protein BPTI Oxytocin has a flexible conformation in aqueous solution and contains no interior water molecules. Because of its small size even its one-dimensional 'H NMR spectrum is well resolved, so that the NOESof the individual polypeptide protons with the water resonance are well separated in 2D NOESY and 2D ROESY experiments." In aqueous solution at temperatures below 6°C the overall rotational tumbling of oxytocin is in the slowmotional regime,22with a rotational correlation time longer than 2 ns. Correspondingly, all NOESY cross-peaks between different polypeptide protons are positive. In contrast, negative NOE cross-peaks between the water line and the peptide signals were observed throughout in a NOESY spectrum recorded at 6"C, showing that the residence times of the hydration water molecules are shorter than 500 ps. The fact that all 'H resonances of oxytocin have negative NOESY cross-peaks with the water line (apart from some positive cross-peaks due to chemical exchange) shows that there are no hydration sites which would be occupied by much more stably bound waters than others. The aforementioned conclusions were confirmed by experiments in the temperature range 0--25 "C, using oxytocin in a mixed solvent of 60 % H20-40 % [2H]acetone.14 At all temperatures, positive NOESY cross-peaks were observed between different peptide protons. In contrast, the sign of the NOE cross-peaks between the water protons
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Protein Hydration in Aqueous Solution
and the protons of the polypeptide is negative above 0 "C and positive at -25 "C. At some intermediate temperature the NOESY cross-peaks vanish, while the intensity of the ROESY cross-peaks increases continuously with decreasing temperature. For different polypeptide protons the sign inversion of aNoE occurs at different temperatures, showing that there are variations of the hydration water residence times in different sites on the protein surface. From these low-temperature experiments there was again no evidence for hydration water that would be bound to oxytocin with a lifetime exceeding 500 ps at temperatures above 0 "C. Based on the experience gained with oxytocin, one might expect that most or all polypeptide protons near the surface of globular proteins should show negative NOESY cross-peaks with the water, So far, in BPTI only a limited number of these expected NOEs have been observed (Fig. 3). We attribute the apparent absence of many of the expected NOESY cross-peaks to the low sensitivity of the experiments used. Most of the negative cross-peaks seen in Fig. 2 and 3 are with intense resonances of BPTI, such as those of methyl groups. This sensitivity criterion applies, however, only to weak NOEs from short-lived hydration waters. Long-lived surface hydration waters would produce much stronger, positive NOESY cross-peaks, comparable to those seen for the internal waters. It is therefore very unlikely that NOEs with surface hydration water molecules bound with residence times >500 ps would have escaped detection by both NOESY and ROESY.
Discussion: Comparison of the X-Ray Diffraction Data on Individual Hydration Water Molecules in BPTI Crystals and the Corresponding NMR Data obtained in Aqueous Solution NMR experiments in solution and X-ray diffraction experiments with protein single crystals are sensitive to different aspects of protein hydration. The sign and intensity of the protein-water NOEs observed by NMR reflect primarily the residence times of the water molecules near the protein protons. In contrast, X-ray diffraction probes the total fraction of time that a water molecule spends at a particular point in space, but is largely insensitive to the residence time at that site on any particular visit.28 On this basis it is of special interest to investigate how the presence or absence of ordered, X-ray observable hydration water in the single crystals can be correlated with the residence times of water molecules in corresponding hydration sites as observed by NMR in solution. BPTI has the same molecular architecture in solution and in crystals, which includes the four internal water molecule^.^-^^^^ In Plate 1the crystal-structure atomic coordinates 5PT13*6were used to generate molecular models, visualizing selected surface properties of the protein in the crystals, and the X-ray and NMR observations on surface hydration. The 'back view' shown is representative of the complete protein surface (the 'front view' was previously presented in ref. 11). With the exception of the protein-protein contact sites in the crystal lattice [yellow in Plate l(a)], the entire protein surface must be covered with hydration water molecules. Only part of the hydration water has so far been observed by either of the two methods considered here. In the BPTI crystal structure, ca. 40 % of the protein surface is involved in protein-protein contacts, 25 % is covered with X-ray-observable hydration waters attributed to the central protein molecule, and 15 % make contacts with X-ray observable water molecules attributed as hydration waters to neighbouring protein molecules [Plate l(b)]. The remaining 20 % of the protein surface must be in contact with water molecules that are not sufficiently well ordered to be seen by X-ray diffraction experiments [comparison of Plate l(a), where white indicates water-accessible hydrogen atoms in the crystal lattice, with Plate l ( b ) , which shows clearly that numerous accessible sites are not covered with X-ray-observable waters). It is further worth noting that more than 40 % of the X-ray-
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(c)
Plate 1. Stereo views of a space-filling CPK representation of the crystal structure 5P"I of BPTI,6 using the following colour codes to visualize salient features of the structure in crystals and in solution. ( a ) Protein surface. Hydrogen, carbon and sulfur atoms are coloured grey, nitrogens blue and oxygens red; hydrogen atoms with more than 20% solvent-accessible surface area in the crystal structure are white and hydrogen atoms within a distance of S 3 . 0 8 , from neighbouring protein molecules in the crystal lattice are yellow. ( b ) Hydration observed in crystals. All polypeptide atoms are grey, the hydration water molecules attributed to this protein molecule in the crystal are green, additional water molecules attributed to hydration sites on neighbouring protein molecules yet located within 3.0 8, are blue-green. (c) NMR observations in aqueous solution. Hydrogen atoms for which 'H-'H NOE cross-peaks with the water resonance were observed are either brown (positive NOESY cross-peaks) or yellow (negative NOESY cross-peaks); polypeptide atom groups with proton chemical shifts at the water resonance, i e . the side-chain hydroxy protons, the N-terminal amino protons and the carboxyl oxygens are coloured magenta; interior hydration waters and the surface-bound water molecule W129, which was observed identically in all three crystal structures5-' (see text) are green; the hydrogen bonding partner of W129, i.e. the amide proton of Ile-19, is red
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observable water molecules are in contact with two protein molecules in the crystal lattice and that all observed waters are in contact with at least one protein molecule, but that most of the observed surface hydration water molecules are also accessible for contact with X-ray unobservable water in the crystal. In aqueous solution all polypeptide hydrogen atoms giving rise to negative cNoE values with the water protons [brown in Plate 1(c)] are located either near the N-terminus, a carboxy group, a hydroxy proton [magenta in Plate l(c)], or near one of the four interior water molecules [green in Plate 1(c)]. From the ensemble of all the experimental observations described in the preceding sections, we arrive at two important points to be made on surface hydration in solution: (i) nearly all positive NOESY cross-peaks between the water resonance and resonances of polypeptide protons on the protein surface are due to the hydroxy protons of serine, threonine and tyrosine, rather than to stably bound water molecules; (ii) the surface hydration sites containing well ordered, X-ray-observable water molecules in the crystal structure have similar residence times for hydration waters in solution as other surface areas for which the hydration water is disordered in the crystals and not observable by X-ray diffraction. Point (i) is supported by the aforementioned experiments in the pH range 5.0-6.5, which showed that almost all of the polypeptide protons near the protein surface that have positive NOESY cross-peaks with the water resonance at more acidic pH give rise to strong NOEs with the hydroxy proton resonances of Ser, Thr and Tyr. Point (ii) results from two independent observations. First, out of a total of ca. 60 X-ray-observable surface hydration waters there are only six water molecules that have conserved hydrogen-bonding partners in all three single-crystal structures of BPTI.5-7 Of those, the hydration sites W143 and W129 [Plate l(c)] could so far be characterized by the NMR data. In the crystal structures, W143 is in hydrogen-bonding distance to the amide proton of Ala-25, which has vanishing cross-peak intensity with the water line in the NOESY experiment and a weak NOE cross-peak in the ROESY spectrum. W129 is within hydrogen-bonding distance of the amide proton of Ile-19, which shows a negative NOESY cross-peak with the water. From these data upper limits for the residence times of
