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Site-resolved measurement of water-protein interactions by solution NMR

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Nathaniel V Nucci1,2, Maxim S Pometun1,2 & A Joshua Wand1,2 The interactions of biological macromolecules with water are fundamental to their structure, dynamics and function. Historically, characterization of the location and residence times of hydration waters of proteins in solution has been quite difficult. Confining proteins within the nanoscale interior of a reverse micelle slows water dynamics, allowing global protein-water interactions to be detected using nuclear magnetic resonance techniques. Complications that normally arise from hydrogen exchange and long-range dipolar coupling are overcome by the nature of the reverse micelle medium. Characterization of the hydration of ubiquitin demonstrates that encapsulation within a reverse micelle allows detection of dozens of hydration waters. Comparison of nuclear Overhauser effects obtained in the laboratory and rotating frames indicate a considerable range of hydration water dynamics is present on the protein surface. In addition, an unprecedented clustering of different hydration-dynamics classes of sites is evident. The interactions of biological macromolecules with solvating water are fundamental to their structure, dynamics and function 1. Historically, comprehensive, site-resolved experimental insight into the behavior of solvent near protein surfaces in solution has been notoriously difficult to obtain. Some years ago, multidimensional solution NMR was used to observe site-resolved dipolar magnetization exchange between protein and water molecules having relatively long-lived interactions2, and this has provided insight into various properties of water intimately involved in determining the structure of proteins3. In principle, the same NMR-based methodology could also allow access to a comprehensive, site-resolved characterization of the interaction of water across the surface of a protein. Unfortunately, this approach has been confounded by the extremely short residence times of hydration water, by complications arising from hydrogenexchange phenomena, and by a potential ambiguity in the distinction between long-range contributions from water molecules in the bulk and those of the hydration waters of interest. It is now well established that the motion of water molecules is somewhat slowed as a result of interaction with the protein surface4. A variety of methods have shown that the one to two layers of water molecules nearest the macromolecular surface are motionally slowed,

though estimates of the degree of retardation vary from several-fold to perhaps two orders of magnitude5–8. Regardless, hydration water– ­protein interactions generally remain sufficiently short-lived to quench the intermolecular dipolar magnetization exchange necessary to detect them in the laboratory using the nuclear Overhauser effect (NOE) or the rotating-frame Overhauser effect (ROE)9. The situation is also considerably clouded by the rapid exchange of solvent hydrogens with labile sites on the protein2,3,9. Furthermore, a more recent theoretical analysis also predicts dominant contributions to the observed NOE/ ROE intensity from long-range intermolecular dipolar couplings 10. This effect arises primarily from two geometric sources that combine to reduce the effective distance-dependence of the NOE and ROE from the familiar inverse-sixth-power dependence to a more slowly varying simple inverse dependence10–12. These restrictions and complications have largely frustrated the exploration of the protein-water interface using high-resolution NMR methods5,13. Here we take advantage of several favorable aspects of reverse micelle encapsulation to circumvent these limitations, enabling us to take site-resolved measurements of the hydration water dynamics for human ubiquitin. RESULTS Encapsulation enables detection of protein hydration water Reverse micelles are monodisperse ensembles of surfactantencapsulated nanoscale water pools that result from spontaneous organization of a mixture of appropriate surfactants, water and non­ polar solvents such as the alkanes (Fig. 1). The size of the water pool can be tightly controlled by varying the molar ratio of water to ­surfactant (water loading, or Wo). Proteins can be encapsulated within the water pool of reverse micelles reproducibly, with high structural fidelity and at concentrations amenable to high-resolution solution NMR ­methods14,15. When encapsulated proteins are prepared in low-viscosity solvents, the full arsenal of solution multidimensional heteronuclear NMR techniques becomes accessible16,17. Reverse micelles have served as a powerful tool for NMR-based studies of protein and nucleic acid structure and biophysics under a range of conditions14,16,18–23. For water loadings such as those used here (W0 ~10), the reverse micelle contains approximately 1,000 molecules on average24, and the reorientational dynamics of encapsulated water is slowed ­relative to bulk water by approximately one order of magnitude25,26.

1Johnson

Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 2Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Correspondence should be addressed to A.J.W. ([email protected]). Received 8 July 2010; accepted 18 October 2010; published online 2 January 2011; doi:10.1038/nsmb.1955

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Figure 1  Schematic of ubiquitin in an AOT reverse micelle. The surfactant, water pool and protein are shown to scale, with a single water molecule pictured, also to scale, for comparison. Nonpolar alkane solvent, which surrounds the reverse micelle particle, is not shown. The diameter of the reverse micelle was determined based on measurements of the rotational correlation time of ubiquitin in AOT reverse micelles at water loading comparable to that used here14. This value agrees closely with simple geometric predictions of the reverse micelle dimensions. The outer, darker blue ring approximates the layer of solvent that is dynamically restricted by interactions with the surfactant headgroups24, whereas the lighter blue region corresponds to the central water pool that hydrates the protein.

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This suggested to us that the interaction with the protein surface ­combined with nanoscale confinement could slow the water dynamics sufficiently to allow efficient detection of hydration water–protein cross-relaxation. At the same time, if reorganization of water contributes to the rate-limiting step, hydrogenexchange chemistry in the reverse micelle should also be somewhat slowed, thereby potentially minimizing complications arising from hydrogen exchange. Finally, reverse micelles also provide an appropriate experimental condition to directly assess the contributions from long-range intermolecular dipolar interactions. We used human ubiquitin to test these ideas. Ubiquitin is a small 76-residue protein comprising a five-stranded mixed β-sheet, a long α-helix and a short 310-helix27. It engages in an extensive set of protein-protein interactions28. We first examined the hydration of ubiquitin in free aqueous solution at pH 5 and 25 °C by conducting three-dimensional 15N-resolved NOESY29 and ROESY30 experiments (Fig. 2a,b). Only a few weak amide-water cross-peaks were observed, which is generally indicative of very short residence times for protein hydration water in bulk aqueous solution3. At this pH and temperature, the exchange of even non–hydrogen-bonded amide hydrogens with solvent is generally too slow to affect these experiments. However, every amide hydrogen-water cross-peak observed in the free-solution spectra involves an amide hydrogen that is within the distance detectable by the NOE (see below) of a labile side chain hydrogen. Chemical exchange of side chain hydrogens with solvent water, followed by intramolecular NOE, produces cross-peaks at the water resonance that are indistinguishable from direct protein-water NOEs2,3. It is likely that many, if not all, of the amide hydrogen– water NOE correlations seen in bulk solution are the result of such exchange-relayed interactions5. We have previously demonstrated that the structure of ubiquitin is unperturbed by appropriate encapsulation in reverse micelles composed of bis(2-ethylhexyl)sulfosuccinate (AOT) 14. In contrast to the paucity of amide hydrogen–water interactions revealed by the NOESY spectrum of ubiquitin in free solution, the protein encapsulated in AOT reverse micelles shows literally dozens of proteinwater interactions under similar temperature and pH conditions (Fig. 2c,d). Dipolar exchange is identified by the opposite phase of 

2.8 Å

the cross-peaks in the orthogonal frames (shown here as positive NOE, negative ROE), whereas direct hydrogen exchange with solvent gives rise to peaks of identical phase (shown as positive in both spectra)2,3. Calibration of the NOE obtained at the 40-ms mixing time used here placed it in the linear regime and corresponds to a maximum NOE-detected distance of approximately 4.3 Å. More than half of detected amide hydrogen-water cross-peaks involve amides that are over 4.3 Å from any labile side chain hydrogen, as indicated by the ensemble of structures determined for encapsulated ubiquitin (PDB code 1G6J)14. These cross-peaks can therefore be unequivocally assigned to direct NOE interactions between the hydration water and amide hydrogens of the protein. In addition, the hydrogenexchange chemistry catalyzed by hydroxide ion within the reverse micelle water pool is effectively slowed by two orders of magnitude, as indicated by the pH dependence of the onset of line broadening due to amide hydrogen exchange (Supplementary Fig. 1). It should be noted that the pKa values of side chain hydroxyls and primary amines are low enough that general base catalysis by the buffer or even by the head groups of AOT is also a potential concern. However, the fact that lysine side chain ζ-amine groups were observed (Fig. 2) and that most side chain hydroxyls could be identified by direct NOE (Supplementary Fig. 2) indicates that there is minimal exchange of hydrogens with solvent. Given the relative concentrations of protein and water, this restricts the hydrogen exchange rates to the 1 s–1 or slower rate regime. The single labile side chain hydrogen that may have a faster chemical exchange rate is the hydroxyl of Tyr59, which is anticipated to have a considerably lower pKa. However, contributions from hydrogen exchange–mediated ­pathways are sufficiently

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Figure 2  Detection of protein hydration by solution NMR. (a–d) 1H-15N planes at the water 1H resonance of three-dimensional NOESY 15N-HSQC (a) and three-dimensional ROESY 15N-HSQC (b) of uniformly 15N-labeled ubiquitin in aqueous solution. In these spectra, the water resonance is at 4.8 p.p.m. Dipolar interactions between amide hydrogen and α-hydrogen (nearly) degenerate with the water resonance are also indicated. Also shown are 1H-15N planes at the water 1H resonance of three-dimensional NOESY 15N-HSQC (c) and three-dimensional ROESY 15N-HSQC (d) of uniformly 1H-15N,2H-12C–labeled ubiquitin encapsulated in AOT reverse micelles at W0 = 9.0. In these spectra, the water resonance is at 4.3 p.p.m. For all panels, positive (black) and negative (red) signs of cross-peak intensity define dipolar (positive NOE, negative ROE) versus chemical exchange peaks (positive NOE, positive ROE). The cross-peaks between amide NH and the water resonance are labeled by residue. All of the cross-peaks observed in the free-solution aqueous spectra arise from amide hydrogens that are within NOE distance of labile side chain hydrogens and are ascribed to hydrogen exchange. In contrast, for encapsulated ubiquitin, only the amide hydrogen of Glu51 (emphasized with an asterisk) is near enough to a rapidly exchanging hydrogen (the hydroxyl of Tyr59) to make a contribution to the NOE cross-peak intensity of more than 2%. Spectra were obtained at pH 5 and 25 °C (aqueous) or 20 °C (reverse micelle) at 500 MHz (1H) using a cryogenically cooled probe.

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minimized to allow quantitative interpretation of the vast majority of the NOE and ROE cross-peak intensities. Interpretation of the NOE/ROE ratio (see below) as directly ­representative of local hydration dynamics also requires that the contributions from long-range dipolar couplings be negligible. NOESY spectra of highly deuterated ubiquitin lack any measurable crosspeaks to organic solvent hydrogens, demonstrating that long-range interactions beyond the exterior of the reverse micelle are minimal. It has been argued that this long-range contribution in bulk aqueous solution provides the vast majority of the NOE intensity to the water resonance10. It is also of note that only a handful of amide hydrogens show NOEs with hydrogens of the surfactant headgroup. With the exception of Gln47 and Gln49, these amide hydrogens are located in the disordered C-terminal tail comprising residues 73–76. The grouping of such interactions suggests that they arise from the local close approach of the protein to the surfactant shell rather than from general long-range dipolar exchange contributions. It is important to note that these regions of the surface are not dehydrated (Fig. 2). The spatial geometry of the reverse micelle and encapsulated protein also causes the number of water molecules to increase roughly as the square of the distance from a solvent-exposed protein hydrogen. This is in contrast to the cubic dependence seen in bulk solution (Supplementary Fig. 3) These fundamental differences in the physical parameters of the reverse micelle system versus bulk solution combine to largely eliminate contamination of short-range dipolar interactions between protein and hydration water by long-range solvent couplings. In summary, the residence times of water on the surface of an encapsulated ubiquitin molecule are sufficiently increased, the effective rates of specific and general base catalysis of hydrogen exchange sufficiently decreased and the potential contribution by long-range coupling to bulk solvent sufficiently reduced to allow confident and comprehensive site-resolved measurements of dipolar cross-relaxation between hydration waters and amide hydrogens of the protein. This allows the first site-resolved analysis of relative hydration water mobilities across an entire protein surface.

The dynamical character of the protein-water interface A quantitative measure of hydration dynamics can be obtained from the ratio of the NOE to ROE cross-relaxation rates, σNOE and σROE (refs. 2,3). Here, ratios of the intensities of the water cross-peaks are used (Supplementary Tables 1 and 2). In the slow correlation time limit (ωτ >>1), a rigidly bound water molecule would result in a NOE/ROE ratio of −0.5 (refs. 2,3). We find that a minority of the resolved proteinwater correlations have ratios at this limit. Previous relaxation studies indicate that the effective macromolecular tumbling time for these reverse micelle particles dissolved in liquid pentane is about 10 ns16. The majority of detected hydration sites showed a range of NOE/ROE values between 0 and −0.5, indicating the presence of substantial motion of the hydration water on the surface of the protein and/or a somewhat shorter residence time3,31. In previous studies of protein-water crossrelaxation, it was unclear to what degree indirect magnetization exchange contributed to NOE/ROE ratios more positive than −0.5. As measurements made in the reverse micelle are generally uncomplicated by indirect exchange effects, we conclude that the range of observed NOE/ROE ratios arises from a distribution of hydration water dynamics that reflects both local geometrical factors and the residence times of the individual water molecules on the surface of ubiquitin31. Approximately three-fourths of the amide hydrogens within the NOE-detection distance limit (4.3 Å) of solvent showed measurable cross-peaks to water. No cross-peaks to water from buried amide hydrogens were detected, confirming the absence of spin-diffusion or appreciable penetration of water into the interior of the protein. A detailed view of the relative hydration dynamics of ubiquitin (Fig. 3) shows clear evidence for clustering of hydration-dynamics classes. A clear grouping of very slow and spatially restricted hydration is evident along the C-terminal tail of the protein, and a cluster of very fast hydration sites is evident along the surface of the α-helix. Clusters of intermediate dynamics are also visible along the mixed β-sheet. These groupings illustrate the relative hydration dynamics essentially across the surface of the entire protein. One noteworthy feature is that a minority of solvent-exposed sites lack detectable cross-relaxation to water. These sites, again clustered along the solvent-exposed ridge of the α-helix, also do not show measurable cross-peaks to surfactant hydrogens, suggesting that long-lived interactions with the inner surface of the reverse micelle shell are absent and that the fastest and least spatially restricted hydration dynamics occur at these amide sites.

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90° 90°

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Figure 3  Ubiquitin hydration dynamics map. All amide hydrogens within NOE distance of solvent are shown as spheres. Sites are color coded as follows: dark blue, bound hydration sites (NOE/ROE ≤ −0.47); cyan, relatively slow hydration (−0.47 > NOE/ROE < −0.3); yellow, relatively fast hydration (−0.3 ≤ NOE/ROE); green, fast hydration (solvent exposed, no water cross-peaks). The dark blue hydration class corresponds to sites where water within NOE distance (4.3 Å) of the amide hydrogen has a residence time on the order of the correlation time of the protein (~10 ns) or longer. Green sites are those that are within NOE distance of solvent, yet show no cross-peaks to water. These represent the fastest sites of hydration dynamics. Images were created using PyMOL (DeLano Scientific)39.

DISCUSSION Until now, experimental efforts to develop a site-resolved understanding of the hydration layer around proteins have relied largely on crystallographic data. Because most protein crystal structures are heavily hydrated and contain complex ensembles of solvent, the general view seems to be that the environment in protein crystals should be representative of the crowded conditions within the cell and that crystallographic water locations should therefore be representative of the native hydration behavior of the protein. Extensive analysis of crystallographic waters has been combined with simulation data in several systems to propose a picture of the molecular nature of the hydration layer32. The present case of ubiquitin serves as a cautionary example. Two crystal structures for wild-type human ubiquitin are available (PDB codes 1UBQ and 1UBI)27,33. Only about half of the defined waters in the two crystal structures were conserved to within 1 Å between the structures, even though the proteins were crystallized in the same space group under almost identical conditions. Furthermore, there is little correlation between the waters detected by solution NMR and the locations and occupancies of the crystallographic waters in these crystal structures (Supplementary Fig. 4). To illustrate the degree of correspondence between the long-lived hydration waters detected by NMR and the locations of the defined waters common to both crystal structures of ubiquitin, a color-coded ribbon diagram of 1UBQ is shown in Figure 4. Notably, only ~60% of the crystallographic waters that are common to both crystal structures appear within the 4.3-Å NOE detection distance of sites where NMRdetected hydration waters are long lived. In addition, more than half of the NMR-detected long-lived protein-water interactions involve amide hydrogens that are outside NOE distance from the nearest conserved crystallographic water molecule. Additionally, we found no correlation between the NOE/ROE values for the NMR-detected sites of long-lived hydration and the location of the nearest conserved crystallographic water. In the case of ubiquitin, there seems to be limited correlation between the locations of the defined water molecules in the two crystal structures and the NMR-detected hydration waters of encapsulated ubiquitin. Indeed, magnetic relaxation dispersion experiments have suggested that a single very long-lived water molecule is present in ubiquitin, which on the basis of the crystal structure (1UBQ) was identified with a water molecule (water 28) localized near the amide NH of Leu43 (ref. 34). Though this water is illuminated by NOEs in encapsulated ubiquitin, its residence time is short enough to cause complete chemical-shift averaging with the 

bulk water resonance. In contrast, encapsulated ubiquitin shows a very long-lived water molecule that is localized to a relatively deep surface pocket and is highlighted by short-distance NOEs between the amide hydrogens of Val69 and Lys6 and a resonance with a chemical shift (5.4 p.p.m.) resolved from the water resonance (4.32 p.p.m.) (Supplementary Fig. 2). These amide hydrogens are not within the NOE detection distance of any hydroxyl group, and we therefore identify this water, which is not resolved in the crystal structure, as the long-lived water molecule suggested by the magnetic resonance dispersion experiments. Notably, this water resides in a pocket that is lined with hydrophobic side chains, which may account for the lack of ordered crystallographic water at this site35. Protein solvation and nanoscale confinement In recent years, there has been an increasing appreciation of the inherent complexity of the cellular context in which proteins must function36. It is now well known that cells are packed with macromolecular surfaces that confine proteins to nanometer-scale volumes. Confinement on this length scale can potentially alter a number of

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Figure 4  Comparison of crystallographic and solution NMR-detected hydration of human ubiquitin. Two views of the backbone ribbon of a crystallographic structure of human ubiquitin (PDB code 1UBQ) are shown. Waters that are common between the two crystal structures (1UBQ and 1UBI) are shown as spheres. The waters are colored according to whether the nearest amide hydrogen does (blue) or does not (green) show NOE and ROE correlations with the water resonance. Buried and unresolved portions of the backbone are colored orange and gray, respectively. Portions of the backbone that are solvent exposed but do not show NOE and ROE cross-peaks to water are colored green, indicating that waters residing at these sites have too short a residence time to be detected. Backbone amide hydrogens that show NOE and ROE crosspeaks to water are colored blue if they are within the NOE detection distance of a conserved crystallographic water or yellow if they are not. Images were created using PyMOL (DeLano Scientific) 39.

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TEC H NICAL RE P ORTS fundamental properties of proteins36 and create an intracellular hydration environment that is potentially highly heterogeneous and distinct from bulk water1. Reverse micelle encapsulation provides a convenient way to undertake detailed studies of the effects of confinement on protein hydration, folding, stability and dynamics on the atomic scale, using solution NMR methods20,37,38. Here we have found a previously unseen range of hydration behavior across the surface of the protein. This represents an important alternate view for the ongoing effort to unravel the interplay between proteins and solvent that is so vital to living systems. Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/nsmb/.

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Note: Supplementary information is available on the Nature Structural & Molecular Biology website. Acknowledgments We are grateful to K. Valentine, J. Gledhill and J. Dogan for helpful discussion and to B. Halle for comments on an early draft of this manuscript. Supported by a grant from the US National Science Foundation (MCB-0842814) and a grant from the Mathers Foundation. N.V.N. is the recipient of a US National Institutes of Health postdoctoral fellowship (GM 087099). AUTHOR CONTRIBUTIONS A.J.W. designed and initiated the study. M.S.P. carried out preliminary NMR experiments. N.V.N. optimized the NMR experiments, and collected and analyzed the data. A.J.W. and N.V.N. wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/nsmb/. Published online at http://www.nature.com/nsmb/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.

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NMR spectroscopy. Uniformly 15N-labeled or 2H,15N-labeled ubiquitin was prepared as described previously40. 2H,15N-labeled ubiquitin in 50 mM sodium acetate buffer, pH 5, with 50 mM NaCl was encapsulated in 75 mM AOT reverse micelles in 99% perdeuterated pentane at W0 = 9.0. Water loading was determined by 1H NMR. For both aqueous and reverse micelle samples, the pH of the sample was confirmed using the pH-dependent 1H chemical shifts of the buffers as internal NMR standards, acetate (pH 5–7) or imidazole (pH 7–9)41. Three-dimensional sensitivity-enhanced42 15N-resolved NOESY-HSQC29,43–45 and ROESY-HSQC30,46,47 spectra were collected at 500 MHz (1H) on a Bruker AVANCE III spectrometer equipped with a cryoprobe. Three-dimensional NOESY and ROESY spectra were collected with 64 (reverse micelle) or 48 (aqueous) and 80 complex increments in the indirect 15N and 1H dimensions, respectively. The ROESY experiment used an 8.33 kHz continuous-wave spinlock field with the 90x-Sly-90x pulse scheme in order avoid spin-lock offset effects48. The mixing time for all three-dimensional experiments was 40 ms with a recycle delay of 1.4 s. Water suppression was achieved using a water-selective sin x/x flip-back pulse after the mixing time. Quadrature detection was achieved by gradient selection and States-TPPI phase cycling in the 15N and 1H indirect dimensions, respectively. NOE/ROE ratios were calculated by fitting the NOESY and ROESY peaks to Gaussian functions with Sparky49 after processing with FELIX (Accelrys). A few weak cross-peaks gave fits with large errors, and maximum peak intensities were used for these sites, as indicated in Supplementary Table 2. In the limit of short mixing time (τmix), the cross-peak intensities are directly representative of the cross-relaxation rates50. 15N T1 (ref. 51) and HzNz (refs. 52,53) relaxation times were measured and found to be long (>500 ms and >100 ms, respectively) compared to the mixing time of the three-dimensional NOESY experiment; thus, autorelaxation effects are minimal in the measured NOE peaks. As a result, the fit volumes of the NOE signals in the water 1H plane (4.32 p.p.m.) were directly proportional to the NOE cross-relaxation rate and were used without additional manipulation. Longitudinal relaxation in the transverse plane (T1ρ) was measured using two-dimensional HSQC experiments for spectral resolution with spin locking as described above for the three-dimensional experiment. Nine mixing times were measured ranging from 2.9 ms to 80 ms, with three of those times measured in duplicate. The measured T1ρ values (Supplementary Table 1) were comparable to the mixing time, and thus the contributions of autorelaxation to the ROE crosspeaks had to be taken into account. NOE/ROE ratios were calculated as shown in Eq. (1), which simply adjusts the intensity of the ROE cross-peaks to account for the attenuation caused by autorelaxation50. −t mix

NOE = ROE

T NOE pv e 1r

ROE pv

(1)

where the subscript ‘pv’ denotes the fitted peak volumes of water cross-peaks from the three-dimensional experiments. Cross-peaks to water were identified by comparing the 15N-resolved NOESY spectrum of encapsulated ubiquitin to that of the protein in free aqueous solution. Calculated NOE/ROE ratios are given in Supplementary Table 2. Structural analysis. Structural analysis of the hydration data obtained in the NOESY and ROESY experiments was done using the reverse micelle ubiquitin NMR structure (PDB code 1G6J). Identification of buried amide hydrogens was done with the Travel Distance Suite54,55, using a 1.4-Å probe to represent water. This water radius was chosen to produce a liberal evaluation of water accessibility to the backbone amides. The minimum distance from each atom to the ­molecular surface was averaged over the 32 structures in the NMR-based ensemble14, and any amide hydrogen that was 4.0 Å or more from the molecular surface was considered to be buried, that is, outside potential NOE-detection distance from water hydrogens. This distance was chosen to account for the 4.3–4.5-Å maximum range of the intramolecular NOEs detected with a 40-ms mixing time. Because the distances calculated were to the Connolly molecular surface56 rather than

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the Lee-Richards solvent-accessible surface57, an additional ~0.5 Å had to be added to account for the effective van der Waals radius of the water hydrogen. Of the eight amide hydrogens that by these criteria were considered buried, three showed cross-peaks to water. The water cross-peaks for Ile3 and Leu56 were weak in both the NOESY and ROESY spectra, suggesting that these sites have more long-range interactions with water. Ile23 showed a strong ROE but a very weak NOE at the water resonance. This indicates that it has a shorter-range interaction with water than other buried residues but the mobility of the water interaction at this site must be quite high. To determine the distances from backbone amide hydrogens to labile ­hydrogens, the interatomic distance was calculated from every backbone amide hydrogen to every labile side chain hydrogen for all 32 structures in the 1G6J ensemble. The distance minimum for each amide hydrogen was averaged over the 32 structures to determine an average minimum amide hydrogen-labile ­hydrogen distance for each amide site. Of the 54 detected protein-water cross-peaks, 32 came from backbone amide hydrogens that had an average minimum distance greater than 4.3 Å from any labile side chain hydrogen To determine how the hydration data are related to the locations of crystallographic water molecules, the two available structures of wild-type human ubiquitin (PDB codes 1UBQ and 1UBI) were compared. Both of these structures were solved using room-temperature X-ray diffraction and share the same space group and the same mother-liquor composition27,33. The protein structures are virtually identical. Following superposition of protein coordinates, waters common to the two structures were identified as those having oxygen coordinate differences of 1 Å or less. Of the 58 waters in 1UBQ and 81 waters in 1UBI, 36 were defined by this criterion as being common to both crystal structures (Supplementary Fig. 4). 40. Wand, A.J., Urbauer, J.L., McEvoy, R.P. & Bieber, R.J. Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein. Biochemistry 35, 6116–6125 (1996). 41. Baryshnikova, O.K., Williams, T.C. & Sykes, B.D. Internal pH indicators for biomolecular NMR. J. Biomol. NMR 41, 5–7 (2008). 42. Palmer, A.G. III, Cavanagh, J., Wright, P.E. & Rance, M. Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy. J. Magn. Reson. 93, 151–170 (1991). 43. Zuiderweg, E.R.P. & Fesik, S.W. Heteronuclear 3-dimensional NMR spectroscopy of the inflammatory protein C5A. Biochemistry 28, 2387–2391 (1989). 44. Bax, A., Ikura, M., Kay, L.E., Torchia, D.A. & Tschudin, R. Comparison of different modes of 2-dimensional reverse-correlation NMR for the study of proteins. J. Magn. Reson. 86, 304–318 (1990). 45. Norwood, T.J., Boyd, J., Heritage, J.E., Soffe, N. & Campbell, I.D. Comparison of techniques for 1H-detected heteronuclear 1H-15N spectroscopy. J. Magn. Reson. 87, 488–501 (1990). 46. Bax, A. & Davis, D.G. Practical aspects of two-dimensional transverse NOE spectroscopy. J. Magn. Reson. 63, 207–213 (1985). 47. Bothner-By, A.A., Stephens, R.L., Lee, J., Warren, C.D. & Jeanloz, R.W. Structure determination of a tetrasaccharide: transient nuclear Overhauser effects in the rotating frame. J. Am. Chem. Soc. 106, 811–813 (1984). 48. Griesinger, C. & Ernst, R.R. Frequency offset effects and their elimination in NMR rotating-frame cross-relaxation spectroscopy. J. Magn. Reson. 75, 261–271 (1987). 49. Goddard, T.D. & Kneller, D.G. SPARKY 3.0. (University of California, San Francisco, San Francisco, California, USA). 50. Macura, S. & Ernst, R.R. Elucidation of cross relaxation in liquids by two-dimensional NMR spectroscopy. Mol. Phys. 41, 95–117 (1980). 51. Farrow, N.A. et al. Backbone dynamics of a free and a phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994). 52. Kay, L.E., Nicholson, L.K., Delaglio, F., Bax, A. & Torchia, D.A. Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins. J. Magn. Reson. 97, 359–375 (1992). 53. Peng, J.W. & Wagner, G. Mapping of spectral density functions using heteronuclear NMR relaxation measurements. J. Magn. Reson. 98, 308–332 (1992). 54. Coleman, R.G. & Sharp, K.A. Travel depth, a new shape descriptor for macromolecules: application to ligand binding. J. Mol. Biol. 362, 441–458 (2006). 55. Coleman, R.G. & Sharp, K.A. Shape and evolution of thermostable protein structure. Proteins 78, 420–433 (2010). 56. Connolly, M.L. Analytical molecular surface calculation. J. Appl. Cryst. 16, 548–558 (1983). 57. Lee, B. & Richards, F.M. The interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379–400 (1971).

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Site-resolved measurement of water-protein ...

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