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NMR Studies of Protein Surface Accessibility

2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês

10.1074/jbc.m107387200

1083-351X

Neri Niccolai, Arianna Ciutti, Ottavia Spiga, Maria Scarselli, Andrea Bernini, Luisa Bracci, Daniela Di Maro, Claudio Dalvit, Henriette Molinari, Gennaro Esposito, Piero Andrea Temussi,

Mass Spectrometry Techniques and Applications

Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity. Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity. one-dimensional two-dimensional bovine pancreatic trypsin inhibitor enhanced protein hydration observed through gradient spectroscopy nuclear Overhauser effect rotating frame Overhauser effect 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl total correlation spectroscopy. FT, Fourier transform free induction decay Interactions of proteins with other molecules can ultimately be ascribed to their surface features. Direct studies of protein surface accessibility are emerging as a new dimension of structural studies of proteins, particularly because repeated observations in either solution (1Liepinsh E. Otting G. Nat. Biotechnol. 1997; 15: 264-268Crossref PubMed Scopus (100) Google Scholar, 2Dalvit C. Floersheim P. Zurini M. Widmer A. J. Biomol. NMR. 1999; 14: 23-32Crossref PubMed Scopus (26) Google Scholar) or crystal state (3Ringe D. Curr. Opin. Struct. Biol. 1995; 5: 825-829Crossref PubMed Scopus (129) Google Scholar, 4Mattos C. Ringe D. Nat. Biotechnol. 1996; 14: 595-599Crossref PubMed Scopus (224) Google Scholar) have pointed out that proteins have regions where small and uncharged organic molecules, even those different from their physiological ligands, preferentially approach the molecular surface and also account for allosteric disruption of substrate binding (5Krantz A. Trends Biotechnol. 1998; 16: 198-199Abstract Full Text Full Text PDF Scopus (4) Google Scholar, 6Savvides S.N. Karplus P.A. J. Biol. Chem. 1996; 271: 8101-8107Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We have shown that these "hot spots" of the protein surface can be easily mapped by a surface survey based on paramagnetic perturbation of conventional NMR spectra (7Scarselli M. Bernini A. Segoni C. Molinari H. Esposito G. Lesk A.M. Laschi F. Temussi P. Niccolai N. J. Biomol. NMR. 1999; 15: 125-133Crossref PubMed Scopus (30) Google Scholar, 8Niccolai N. Spadaccini R. Scarselli M. Bernini A. Crescenzi O. Spiga O. Ciutti A. Di Maro D. Bracci L. Dalvit C. Temussi P.A. Protein Sci. 2001; 10: 1498-1507Crossref PubMed Scopus (56) Google Scholar). The surface properties are dictated by the relative position and specific features of exposed residues, but even detailed knowledge of the protein architecture may not be sufficient for a thorough description of surface properties because of the intrinsic disorder of these residue side chains. The complex properties of the protein surface are modulated by a variety of factors (e.g.electrostatics, hydrophobicity, and hydrogen bond ability) but share a common unifying feature: hydration. The blanket of water covering the protein surface is the actual interface between the solution environment and the underlying modulations. The possibility of exploiting the blanket resides mainly on two of its features, namely, the variable thickness of the water layers and the fact that residence times of water molecules vary from point to point (9Brunne R.M. Liepinsh E. Otting G. Wüthrich K. van Gunsteren W.F. J. Mol. Biol. 1993; 231: 1040-1048Crossref PubMed Scopus (209) Google Scholar). Since the pioneering studies on protein hydration by Wüthrich's and co-workers (10Otting G. Wüthrich K. J. Am. Chem. Soc. 1989; 111: 1871-1875Crossref Scopus (301) Google Scholar, 11Otting G. Liepinsh E. Wüthrich K. J. Am. Chem. Soc. 1991; 113: 4363-4364Crossref Scopus (101) Google Scholar), it has been well established that NMR is a reliable technique with which to detect water molecules bound to proteins (12Otting G. Prog. NMR Spectrosc. 1997; 31: 259-285Abstract Full Text PDF Scopus (206) Google Scholar). The early approaches were burdened by delicate hardware requirements, but now, thanks to developments in gradient-controlled sequences (12Otting G. Prog. NMR Spectrosc. 1997; 31: 259-285Abstract Full Text PDF Scopus (206) Google Scholar, 13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar, 14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar, 15Melacini G. Boelens R. Kaptein R. J. Biomol. NMR. 1999; 15: 189-201Crossref PubMed Scopus (13) Google Scholar, 16Melacini G. Kaptein R. Boelens R. J. Magn. Reson. 1999; : 214-248Crossref PubMed Scopus (16) Google Scholar), intermolecular nuclear Overhauser effects between water and protein molecules can be routinely measured and correlated to overall protein hydration. It has recently been proposed that the ability of surface mapping to reveal hot spots on the protein surface relies on the hindrance of bound water molecules to the approach of suitable paramagnetic probes to the protein (7Scarselli M. Bernini A. Segoni C. Molinari H. Esposito G. Lesk A.M. Laschi F. Temussi P. Niccolai N. J. Biomol. NMR. 1999; 15: 125-133Crossref PubMed Scopus (30) Google Scholar). A combined use of paramagnetic perturbation and water-protein Overhauser spectroscopy based on 1D1 and 2D ePHOGSY (13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar, 14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar) can prove this link and may thus yield a faithful description of the static and dynamic properties of the water blanket. The attenuation of a specific set of hydrogen resonances in the NMR spectrum in the presence of a soluble paramagnetic probe reflects the accessibility of the corresponding locations on the protein surface (17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar). On the other hand, ePHOGSY experiments trace out different types of water-protein interactions by means of an initial intermolecular magnetization transfer step due to chemical exchange or dipolar interactions or both processes. Provided some prior knowledge of the protein structure (which is necessary anyway for result interpretation) is available, coupling of the two techniques should enable one to characterize the solvent modulation of the protein surface accessibility. Experimental assessment of different "modes" of water molecules on the protein surface can explain subtle phenomena that are difficult to reveal even by sophisticated molecular dynamic simulations because of the still approximate nature of available force fields. BPTI and tendamistat, two paradigmatic proteins that are well characterized both structurally (18Wlodawer A. Nachman J. Gilliland G.L. J. Mol. Biol. 1987; 198: 469-480Crossref PubMed Scopus (196) Google Scholar, 19Berndt K.D. Guntert P. Orbons L.P. Wüthrich K. J. Mol. Biol. 1992; 227: 757-775Crossref PubMed Scopus (184) Google Scholar, 20Pflugrath J.W. Wiegand G. Huber R. Vertesy L. J. Mol. Biol. 1986; 189: 383-386Crossref PubMed Scopus (179) Google Scholar, 21Billeter M. Schaumann T. Braun W. Wüthrich K. Biopolymers. 1990; 29: 695-706Crossref Scopus (56) Google Scholar) and with respect to interactions with the paramagnetic probe 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPOL), represent an ideal benchmark to test this novel surface survey approach. Tendamistat (a gift from Hoechst AG) and BPTI (obtained from Fluka) were purified by high pressure liquid chromatography, whereas TEMPOL (obtained from Sigma) was used without any further manipulation. Tendamistat and BPTI samples were prepared by dissolving the proteins in H2O/2H2O (90:10, v/v) to make 2 and 6 mm solutions, respectively, adjusted to pH 3.15 and 3.5. The paramagnetic NMR samples contained an optimal TEMPOL concentration, which was achieved at a nitroxide/protein ratio of 10:1. This condition was reached by adding a few microliters of a 2 m TEMPOL solution directly to the NMR tube. NMR measurements were obtained at 309 and 323 K, respectively, for BPTI and tendamistat with a Bruker Avance 600 spectrometer to reproduce the experimental conditions of the original structural studies (19Berndt K.D. Guntert P. Orbons L.P. Wüthrich K. J. Mol. Biol. 1992; 227: 757-775Crossref PubMed Scopus (184) Google Scholar, 21Billeter M. Schaumann T. Braun W. Wüthrich K. Biopolymers. 1990; 29: 695-706Crossref Scopus (56) Google Scholar). Data processing was performed with the Bruker software, and the spectral analysis was performed with NMRView (24Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2648) Google Scholar). The 1H chemical shifts were referenced on trimethylsilylpropionic 2,2,3,3-d4 acid sodium salt at 0 ppm. Resonance assignments for both proteins, which were available from the original structural work (19Berndt K.D. Guntert P. Orbons L.P. Wüthrich K. J. Mol. Biol. 1992; 227: 757-775Crossref PubMed Scopus (184) Google Scholar, 21Billeter M. Schaumann T. Braun W. Wüthrich K. Biopolymers. 1990; 29: 695-706Crossref Scopus (56) Google Scholar), were checked anew under our experimental conditions using the scheme of sequential assignment described by Wüthrich (25Wüthrich K. NMR of Proteins and Nucleic Acids. J. Wiley and Sons, New York1986: 130-161Google Scholar). According to this scheme, a conventional set of 2D spectra was recorded: correlated spectroscopy (26Aue W.P. Bartholdi E. Ernst R.R. J. Chem. Phys. 1976; 64: 2229-2246Crossref Scopus (3076) Google Scholar), TOCSY (27Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar), and nuclear Overhauser enhancement and exchange spectroscopy (28Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4809) Google Scholar). Water resonance was attenuated using a DANTE presaturation train superimposed on the specific sequence. Additional experimental parameters were the same as those described previously (7Scarselli M. Bernini A. Segoni C. Molinari H. Esposito G. Lesk A.M. Laschi F. Temussi P. Niccolai N. J. Biomol. NMR. 1999; 15: 125-133Crossref PubMed Scopus (30) Google Scholar,17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar). A total of 470 increments were collected in t1, with 1908 data points and 64 scans/FID in t2, over a spectral width of 6 kHz in both dimensions. Before 2D FT, the experimental array was zero-filled to a final matrix of 2048 × 1024 data points. 1D ePHOGSY was performed with NOE and with ROE to discriminate between Overhauser and exchange effects, and 2D ePHOGSY-NOE-TOCSY (13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar,14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar) was performed for a quantitative analysis of the effects. In all 1D ePHOGSY experiments, spectra were acquired with 16,384 data points and 4096 scans, whereas the 2D ePHOGSY-NOE-TOCSY spectra were obtained with 256 increments and 320 scans over 2048 t2 data points. After zero-filling, the final matrix was 4096 × 512 data points. In all ePHOGSY experiments, the mixing time to build up the intermolecular Overhauser effects was 200 ms, and the H2O-selective 180° Gaussian pulse between the first two pulsed field gradients had a duration of 50 ms and 60 db attenuation (13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar, 14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar). Water suppression was achieved through an excitation sculpting (29Hwang T.L. Shaka A.J. J. Magn. Reson. 1995; A 112: 275-279Crossref Scopus (1511) Google Scholar) module appended to the sequences (13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar, 14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar). The experimental conditions of TOCSY, nuclear Overhauser enhancement and exchange spectroscopy, and 1H-13C heteronuclear single quantum coherence spectra have been described elsewhere (7Scarselli M. Bernini A. Segoni C. Molinari H. Esposito G. Lesk A.M. Laschi F. Temussi P. Niccolai N. J. Biomol. NMR. 1999; 15: 125-133Crossref PubMed Scopus (30) Google Scholar, 17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar). The decrease of peak intensities caused by the added TEMPOL was evaluated from a comparison of ePHOGSY-TOCSY with NOE spectra obtained in the presence and absence of the paramagnetic probe and performed and processed with the same parameters. As reported previously (17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar), paramagnetic effects were measured by comparing autoscaled cross-peak attenuation figures (Ai, paramagnetic attenuation) defined as: Ai=2−(Vip/Vid)Eq. 1 i.e. the individual deviations from the average of the cross-peak autoscaled volumes, Vip,d, the latter of which is defined as:Vip,d=Vip,d/(1/n)(ΣiVip,d)Eq. 2 where n is the number of measured cross-peak volumes, and Vid and Vip are the protein individual cross-peak volumes measured in the absence and presence of the spin probe, respectively. The individual Ai values are plotted versusprotein sequence position, and the values above or below the average attenuation level (unitary by construction because (Σi Vip,d/n) = 1) correspond to high or low spin probe accessibility levels, respectively (17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar). With this representation, it is easy to compare experiments performed under different conditions (temperature, protein and paramagnetic probe concentration, and solvents) because any specific effect is included in the mean value and the observed attenuations. A reference BPTI model was built by fitting all the backbone atoms of the solution and crystal structures found in the Protein Data Bank (Protein Data Bank code 1PIT and 1BPI, respectively). The best fit (root mean square deviation = 0.728 Å) was achieved by comparing the fifteenth structure of 1PIT with 1BPI. In the hydrated solution structure, the coordinates of the four buried water molecules W111, W112, W113, and W122, according to the nomenclature of the crystallographic Protein Data Bank file 6PTI, were incorporated together with W105, W110, W138, and W158, conserved in all the available BPTI x-ray data. The reference tendamistat model was built by fitting all the backbone atoms of the solution and crystal structures of the Protein Data Bank (Protein Data Bank code 4AIT and 1HOE, respectively) with a root mean square deviation = 2.22 Å. Then, the coordinates of the water molecules W84 and W85, according to the numbering of the crystal structure, were added to the solution structure. These waters, which are indeed partially buried and well conserved in the available crystal structures1HOE and 1BVN, should be considered structural ones. Both models were refined with 500 steps of energy minimization by the steepest descent method. The resulting structures were placed in a truncated octahedral box of equilibrated water. Ten steps of energy minimization with the steepest descent method followed by another 490 steps with the conjugate gradient method were carried out. Afterward, the equilibration of the BPTI/water and tendamistat/water systems was done in three stages by a molecular dynamic approach at increasing temperatures. In all runs, the temperatures of the protein and solvent were separately coupled to an isothermic bath. The pressure was kept constant by coupling to an isobaric bath at 1 bar. Bonds were always kept rigid using the SHAKE method with a relative tolerance of 10−4 Å. The length of the molecular dynamic elementary time step was 0.002 ps in all runs. During the first 2 ps trajectory at 100 K, the constants for coupling to the temperature and the pressure bath were 0.05 ps. For the subsequent 2 ps, the temperature was increased to 200 K. Finally, a run of 6 ps at 277 K was performed with temperature and pressure equilibration constants increased to 0.1 and 0.5 ps, respectively. All displayed structures derived from the above-mentioned protocols were generated with the program MOLMOL (30Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 29-32Crossref Scopus (6454) Google Scholar). In Fig. 1, the relative signal intensities measured in 2D ePHOGSY-NOE-TOCSY spectra (13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar, 14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar) of BPTI and tendamistat are reported with bar heights referring to the sum of the intensities of the cross-peaks measured in the ω2dimension. Sizeable water-protein Overhauser effects arising from intermolecular contacts shorter than 4–5 Å report on direct interactions between the solute and water molecules in its first hydration shell (12Otting G. Prog. NMR Spectrosc. 1997; 31: 259-285Abstract Full Text PDF Scopus (206) Google Scholar); hence, the number of the observed water-protein NOEs should be, at first approximation, proportional to the protein accessible surface. This is not the case for the two proteins here investigated. The models built by using Protein Data Bank 4AIT and 1PIT entries, corresponding respectively to hydrated solution structure of tendamistat and BPTI, indicate that tendamistat has a water accessible surface of 4699.2 Å2, i.e. 11% larger than that of BPTI. Furthermore, in agreement with the different number of amino acid residues of the two proteins, BPTI exhibits 328 resolved proton signals, as compared with the 391 signals of tendamistat. In principle, both of these features should lead to a larger number of ePHOGSY signals for the latter protein. On the contrary, 121 and 86 protons of BPTI and tendamistat (37% and 22%, respectively, of the corresponding resolved NMR protons) yield ePHOGSY signals. This observation might reflect, in part, the difference in temperature of the data collection in the two structure determinations. However, the complex nature of the water blanket covering the two surfaces plays a mayor role (vide infra). ePHOGSY spectra report on protein hydrogen connectivities to water via all sorts of magnetization transfer effects, including the direct chemical exchange with bulk water that involves side chain or backbone labile protons. By comparing the peak sign of 1D ePHOGSY-NOE and ePHOGSY-ROE, direct information on the contributions from different magnetization-transfer time scales can be retrieved (13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar, 14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar). Thus, the intensities of the peaks arising from protons undergoing chemical exchange are positive in the 1D ePHOGSY-ROE (Fig. 2), i.e. opposite to any Overhauser effects in the rotating frame. Instead, in 1D ePHOGSY-NOE, the intensity of the water-protein Overhauser effects, which depends on the length and reorientation rate of the specific intermolecular internuclear vector, most commonly has the same sign as the chemical exchange effects as a consequence of the low frequency motional regime of macromolecules (negative Overhauser effect). It should be noted that both these mechanisms could contribute at the same time to the observed intensity of an individual ePHOGSY signal. From inspection of the histogram heights of Fig. 1, it is apparent that the exchange signals found in the 1D ePHOGSY-ROE are among the peaks with the highest intensity. Indeed, several protons of both proteins appear to be involved in chemical exchange processes. This is the case for tendamistat amide protons within the active site, i.e. S17, R19, and Y20, and for several other hydrogens highlighted in the same figure. However, according to previous observations (12Otting G. Prog. NMR Spectrosc. 1997; 31: 259-285Abstract Full Text PDF Scopus (206) Google Scholar), all ePHOGSY peaks of serines, threonines, and tyrosines could be partly due to relayed transfers via the exchanging OH groups of these residues. This could partially explain the very high intensity of ePHOGSY signals of Y10 Hδ and Hε of BPTI and, in general, the enhancement observed in both proteins for any of the three OH-bearing residues. Because of the small size of the proteins, the high temperature, and the short mixing time used in the experiment, we can safely confine the chemical exchange contribution to only the protons in direct contact with the exchangeable protons. Almost all the detectable water-protein NOEs are negative (corresponding to positive peaks in Fig. 2), but a few positive Overhauser effects can be observed, both in the 1D ePHOGSY and 2D ePHOGSY-TOCSY spectra with NOE of BPTI and tendamistat. In the case of BPTI, positive effects are observed for L6 Hγ, A16 Hα, I19 Hβ, K26 Hε, and L29 Hβ3, all nuclei belonging to aliphatic moieties located on the surface of the protein. This feature may suggest that local high flexibility is responsible for this NOE sign inversion. Water molecules bound to surface hydrogens with residence times consistent with the build up of sizeable NOEs and involved in intermolecular dipolar interactions with mutual reorientation faster than 3 × 10−10 s (the sign inversion limit at the operative Larmor frequency) should indeed give positive Overhauser effects. A similar pattern is found for tendamistat because positive NOE effects (negative ePHOGSY peaks) are detected for the surface-exposed aliphatic side chain protons T2 Hγ, T3 Hγ, V4 Hγ, and L44 Hδ, as shown in Fig. 2 b. However, some care must be exerted before ascribing such results exclusively to the local flexibility of the macromolecules. Although positive NOEs between water and a structured macromolecule can always be considered as true dipolar effects, the same outcome would also be expected if the local mobility were due to bound but locally reorienting water molecules or to rapidly diffusing ones (12Otting G. Prog. NMR Spectrosc. 1997; 31: 259-285Abstract Full Text PDF Scopus (206) Google Scholar). Additional independent measurements, such as NMR relaxation or diffusion-filtered determinations, are necessary to safely attribute the local mobility to the macromolecule rather than water or some combination of individual motions. Once the characteristics of all the peaks found in ePHOGSY-ROE and ePHOGSY-NOE spectra are analyzed in detail, and Overhauser effects and exchange processes are properly recognized, possibly by extending the experimental data set with diffusion-filtered determinations (13Dalvit C. J. Magn. Reson. B. 1996; 112: 282-288Crossref PubMed Scopus (47) Google Scholar, 14Dalvit C. J. Biomol. NMR. 1998; 11: 437-444Crossref Scopus (129) Google Scholar), it is possible, in principle, to interpret the NOE intensities in terms of internuclear distances and/or molecular dynamics, but such a calculation is far from trivial. Apart from possible difficulties with overlap of NOEs and relayed exchange contributions, even for genuine negative NOEs, no a priori assumption on correlation times or interproton distance calibrations can be easily made, as is customarily done with intramolecular Overhauser effects. On the other hand, when trying to map the features of the overall accessibility to a protein surface, we are more interested in relative mobility than in absolute mobility. Thus, in this report, no absolute analysis of the ePHOGSY peak intensities has been performed, and only the corresponding TEMPOL-induced paramagnetic perturbations are discussed. Additions of TEMPOL to the water solutions of BPTI and tendamistat cause, to different extents, attenuations of the 2D ePHOGSY-NOE-TOCSY peaks, in total analogy with the effects induced by the same spin label in other more conventional 2D spectra (17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar). Attenuations of 2D ePHOGSY-NOE-TOCSY peaks filter out the set of protein hydrogens whose accessibility is mediated by neighboring water molecules, whereas the corresponding attenuations in conventional 2D spectra (7Scarselli M. Bernini A. Segoni C. Molinari H. Esposito G. Lesk A.M. Laschi F. Temussi P. Niccolai N. J. Biomol. NMR. 1999; 15: 125-133Crossref PubMed Scopus (30) Google Scholar) yield the global accessibility. A comparison between the two ensembles allows the definition of fine aspects of accessibility. TEMPOL-edited ePHOGSY intensities, in fact, enable one to classify the protein-connected water molecules as: 1) bound (dynamically bound) to exposed and flexible (rigid) protein locations, 2) bound to exposed and rigid protein locations, 3) buried, or 4) ordered in a network that prevents close approach of small uncharged ligands. However, recognition of chemical exchange interactions that do not concern the entire water molecule but only its protons still rests on the comparison of ePHOGSY-ROE and ePHOGSY-NOE peak signs. Fig. 3 shows the comparison of 2D ePHOGSY-NOE-TOCSY peak intensities measured in the presence and absence of the paramagnetic probe. Autoscaled peak volumes of all detected signals are compared, as suggested previously (17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar) for comparisons involving different data sets. The signal intensities of both proteins are modulated by specific attenuations along the sequence, but it is apparent that, overall, BPTI ePHOGSY signals are much less attenuated by TEMPOL than those of tendamistat. This finding is fully consistent with the presence of the four water molecules buried in the BPTI interior (18Wlodawer A. Nachman J. Gilliland G.L. J. Mol. Biol. 1987; 198: 469-480Crossref PubMed Scopus (196) Google Scholar, 19Berndt K.D. Guntert P. Orbons L.P. Wüthrich K. J. Mol. Biol. 1992; 227: 757-775Crossref PubMed Scopus (184) Google Scholar). These water molecules can yield an efficient intermolecular magnetization transfer to the inner proton environment of the protein that cannot be perturbed by the external paramagnetic probe. This situation is well illustrated by Fig. 4 a, which shows a BPTI three-dimensional model in which the internal waters (highlighted) are surrounded by all the hydrogens within a 5 Å shell from the oxygen atom of each of the buried waters. It is clear that resonances of protons "illuminated" by buried water magnetization cannot be quenched by TEMPOL. Most of the BPTI ePHOGSY signals exhibiting very low TEMPOL-induced attenuations are located in the inner part of the protein. In fact, 19 of 35 ePHOGSY signals that exhibit Ai < 0.4, an already proposed upper limit for unperturbed signals (7Scarselli M. Bernini A. Segoni C. Molinari H. Esposito G. Lesk A.M. Laschi F. Temussi P. Niccolai N. J. Biomol. NMR. 1999; 15: 125-133Crossref PubMed Scopus (30) Google Scholar), are related to hydrogens that have close contacts with the internal waters. The remaining 16 hydrogens with Ai < 0.4 can be divided in two groups. R1Hδ3, F4NH, Y23Hδ, Y23Hε, Q31Hγ3, Y35Hδ1, Q31Hβ3, and T54Hβ, although not completely buried, are sterically hindered with respect to the approach of a paramagnetic probe of the size of TEMPOL. Others, however, are surface hydrogens, i.e. Q31Hγ1, R39Hα, E49Hβ, C55Hα, and A58Hα, that are totally unhindered not only when viewed in a static picture but also in molecular dynamics simulations. The analysis of the reduced accessibility of these hydrogens is less straightforward, but it discloses a new scenario with respect to previous views of the surface of BPTI. The most direct explanation is the presence of water molecules with long residence times in close proximity to the poorly attenuated surface hydrogens. The presence of many hydrogen bond donor and/or acceptor groups near the latter nuclei is consistent for an ordered network of water molecules that prevents the close approach of TEMPOL to these surface sites.Figure 4Hydrogen environment of buried water molecules: three-dimensional models in which buried waters (highlighted) are surrounded by all the hydrogens within a 5 Å distance (a, BPTI ; b,tendamistat) are shown. In both cases, atoms are colored according to their paramagnetic attenuation (Ai) (see color bar). Gray refers to atoms whose missing Ai value is due to NOE absence, missing assignment, or spectral overlapping. The lack of detectable NOEs is observed only for hydrogens that are more than 4 Å distant from the water oxygen atom.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Only 7 tendamistat ePHOGSY signals exhibit paramagnetic attenuations < 0.4. The fact that this number of signals is very limited can be ascribed primarily to the absence of totally buried water molecules. However, water molecule W84 (according to the 1HOEwater numbering) buried in our reference model and conserved in 1HOEand 1BVN structures accounts for the low attenuations of K34Hα, V35NH, L44NH, and V56Hβ ePHOGSY signals (see Fig. 4 b). It is worth noting that the other three hydrogens whose Ai is 1, i.e. medium to strong paramagnetic effects arising from close encounters of these hydrogens with both water and TEMPOL molecules. The high selectivity attainable by ePHOGSY experiments in connection with paramagnetic perturbations is best exemplified by the possibility of explaining even minute discrepancies between expected and predicted accessible surface areas. The paramagnetic perturbation profile of the1H-13C heteronuclear single quantum coherence spectrum of BPTI was obtained for only 37 resolved heteronuclear correlations Cα-Hα relevant correlations of individual α protons (17Molinari H. Esposito G. Pegna M. Ragona L. Niccolai N. Zetta L. Biophys. J. 1997; 73: 382-396Abstract Full Text PDF PubMed Scopus (36) Google Scholar). The agreement between the paramagnetic attenuation (Ai) and accessible surface areas of BPTI α protons reported in the quoted paper was good in 24 cases. The remaining 13 discrepancies came from residues P2, F4, R39, K41, R42, and T54, whose Cα-Hα correlations exhibited paramagnetic attenuations smaller than the expected ones, whereas residues T11, C14, K15, R17, L29, V34, and G36 exhibited Ai values > accessible surface areas. This finding reflects the fact that the paramagnetic perturbation profiles are not simply related to the static hydrogen topology inferred from the protein structure. A direct comparison of the exposed surface areas of hydrogens of the various residues with the paramagnetic attenuations measured both in ePHOGSY and1H-13C heteronuclear single quantum coherence spectra yields information on differential accessibilities of the protein surface. Thus, the presence of tightly bound water molecules that prevent the approach of TEMPOL to BPTI can explain the Ai values observed for residues F4, R39, K41, R42, and T54 because they have one Overhauser effect with water, at least. In the case of P2, which is very close to the very hydrated F4, an indirect shielding from the paramagnetic probe could be effective. It is interesting to note that as shown by Fig. 5 a, all the residues with anomalous low attenuations are aligned on the same side of the protein surface. From Fig. 5 b, it is also apparent that residues T11, C14, K15, R17, V34, and G36, which are characterized by anomalously high attenuations, define a surface region centered on the protein active site. It is fair to conclude that an overall picture of protein surface accessibility, valid at least for the two chosen examples, is obtained by the combined use of the TEMPOL-induced paramagnetic perturbations on the 2D conventional spectra and the ePHOGSY or some other equivalent spectroscopy experiment. Thus, most of the water molecules involved in close contacts with proteins appear to be characterized by residence times short enough to prevent the onset of detectable Overhauser effects. Tightly bound water is never found near the protein active sites, which always result to be very TEMPOL accessible. This feature is of primary relevance because in this way it is experimentally confirmed that active sites are molecular regions that are very accessible by chemical species, even those that are quite different from the natural ligand. Almost all the surface-exposed regions showing NOE with water are also TEMPOL accessible, unless the solvent molecules are involved in the formation of strong hydration sites. Structural water molecules, which, in general, are buried and thus not at all perturbed by the paramagnetic probe, can be easily identified. It can be concluded, therefore, that from paramagnetic attenuation profiles of protein NMR spectra with and without water-protein dipolar-interaction editing, many critical characteristics of protein surface accessibility may be obtained. Thus, provided that the protein structure, (possibly in solution) is known, a new dimension of structural biology given by the protein accessibility can be explored. Detailed knowledge of all features of the protein surface is of utmost importance in all biological problems involving molecular recognition. As mentioned in the "Introduction," a study of the surface of MNEI, sweet protein, by means of TEMPOL revealed the presence, on its surface, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis (8Niccolai N. Spadaccini R. Scarselli M. Bernini A. Crescenzi O. Spiga O. Ciutti A. Di Maro D. Bracci L. Dalvit C. Temussi P.A. Protein Sci. 2001; 10: 1498-1507Crossref PubMed Scopus (56) Google Scholar). If we limit the discussion to the two main examples of this study, it can be said that a better understanding of the borders and properties of the surface areas of inhibitors can lead to the rational design of mutants with tailored specificity, a vital need in the case, for instance, of viral protease inhibitors. Obviously, other paramagnetic probes might be used, particularly in the case of redox protein systems, which can be affected by the weak oxidant properties of TEMPOL. We thank Hoechst AG for the kind gift of tendamistat.