J. Phys. Chem. 1992,96, 7157-7159
7157
A Microscopic View of Protein Solvatlon VnEe Lounnas, B. Montgomery Pettitt,*it L.Findsen,* and S . Subramaniami Chemistry Department, University of Houston, Houston, Texas 77204-5641 (Received: May 26, 1992; In Final Form: July 24, 1992) Because of difficulties associated with experimental measurement techniques, the distribution of water density around many proteins is not well-resolved. We present, in this paper, a molecular dynamics approach to the general problem of comparing the instantaneousvs average view of protein hydration via a 150-ps simulation of metmyoglobin in an explicit aqueous environment. Densities as a function of position for both water and myoglobin were computed by timeaveraging the volume fraction occupied at differentpositions in space. The picture so obtained challenges the view of hydration taken from accessible surface features related to the average structure. A detailed picture of protein hydration is given that includes significant surface penetration and transient channels, in conjunction with the accepted concepts of a tightly bound partial layer of water on the surface near charged groups.
Our current conceptions of protein hydration, derived mostly from diffraction' and some NMR experiments? have a few water molecules tightly bound near exposed polar residues and tucked away in small interior pockets. The majority of the waters of r o t e n i q t a l s 3 are essentially hydration in all but the driest, small p invisible to these experimental techniques that have revealed so much information about the structure of the proteins themselves.' This is due to the diffusive mobility of water as a liquid wen when the immersed proteins form a regular crystal lattice. For protein structures with common resolution (1.5-3.0 A) and typical R factors after refinement, features with equilibrium populations less than 20-3096 are usually lost or uninterpretable. Computer experiments of proteins and solutions are capable, in principle, of revealing great detail regarding the inherent structures and dynamics available to such system^.^ Simulations are limited by the accuracy of the force field and the accessible time scales. Nonetheless, they have challenged and stimulated our conceptions about proteins and are capable of demonstrating the reproduction of many experimentally accessible quantities regarding structure.6 A theoretical tool that has given insight into the patterns of hydration of proteins is the concept of solvent-accessible surface area.' Using the available crystallographicdata base8,S numerous studies of the solvent-exposed regions of proteins have been performed! Such work has demonstratedcorrelations of accessible surface area with many other hydration-dependent properties. In thu paper, we report a comparison of the average vs instantaneous views of protein hydration obtained by a computer simulation of metmyoglobin in water, which emphasizes the importance of timedependent fluctuations in structure. The initial coordinates for myoglobin, obtained from X-ray crystalIography,'O included 83 waters of hydration. To this, another 3045 water molecules were added to produce a box of 56.32 A by 56.32 A by 44.45 A with no water closer than 2.7 A to protein atoms. The SPC" water model and Amber protein force fieldI2 were used as potential functions. An interaction cutoff of 7 A and a time step of 2 fs were employed. After some minimization to relieve bad contacts, the system was heated to 300 K over 15 ps and equilibrated for another 14 ps, after which a 150-pssimulation in the NYT ensemble followed. Other details and structural analysis of this simulation have been previously given." The time-averaged density of water (volume normalized) in a 2-A-thick plane near the center of the protein is shown in Figure 1 as evenly spaced contours from 5% to 200%of the density of bulk water. The density was computed in bins (0.5 A)3. The water molecules near acidic or basic side chains are most readily recognized as prominent peaks. These waters of hydration are es*To whom correspondence should be addressed. 'Alfred B. Sloan Fellow, 1989-1991. *Present a d d m : Department of Medicinal and Biological Chemistry, University of Toledo, Toledo, OH. 'Resent address: Department of Biophysics, University of Illinois, Urbana, IL.
sentially those expected to be revealed by the aforementioned experimental techniques as belonging to the solvation shell of the protein. The presence of water molecules in the classical ligand entry channel and at the back of the binding pocket is also evident near the contour label 3.14 In Figure 2, we show a region of the protein where the timeaveraged protein density (dot contours) is superimposed on the water density. The most striking feature apparent is that the boundary for the protein solvent interface is not at all sharply defined. Water density appears within the outer border of the protein interpenetrating 3-5 A. Detailed inspection reveals that atomic fluctuations on the surface of the protein, involving mostly side chains, are responsible for smoothing the separation between water and protein and allow some mobile water molecules to move freely into and out of the short-lived crevassa. In addition, global breathing modes evident in the radius of gyration" also contribute to this phenomenon to a lesser extent. Figure 3 shows a onedimensional representation of the number (singlet) density of atoms for both protein and water through a section of the sample. At low percentage values of density significant penetration is found; over 30% little is found. Notice that the classical waters of hydration near the charged surface groups appear as strong features in the water density, essentially outside of the lowest protein contours. The complicated instantaneous structures produced by penetration of water molecules are not adequately characterized by the solvation shell picture typical for small By considering the protein density probability distribution (as a function of p i t i o n ) a fled condition, one may also then relate the water density to the anisotropic protein-water pair probability distribution function.I6 The gradual rise of the water correlations until the protein surface is reached, in Figure 3, is not related to the rise seen in isotropic or angle-averaged pair correlations of proteins and peptides in water." That effect is primarily due to the excluded volume of the solute as seen from a random offcenter site averaged over all orientations. The penetration seen in the anisotropic correlationspresented here would be essentially washed out by the angle-averaging procedure. Some of the information seen in these density distribution functions is apparently accessible in crystallographic experiments. Attempts have been made to refine aspects of the essentially continuous probability distribution of mobile waters surrounding protein structures.17 Such refinement yields solvent structure which has some features in common with the detailed distributions obtained here, includw the characteristicliquid structure rippling over several diameters. Such behavior is expected for water in such confined places. The fact that average proteinsolvent boundaries may not be distinct or simply definable may have implications for a number of other endeavors. As alluded to above, some information on solvent is accessible in current experiments. It should be possible to use the information on the continuous solvent distribution around a protein available from simulation to aid in refinement.
0022-365419212096-7157$03.00/0 @ 1992 American Chemical Society
7158 The Journal of Physical Chemistry, Vol. 96, No. 18, 1992 0.6
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purely numerical technique may now be extended and given physical justification. Our picture of protein solvation suggests that the dielectric tapering can be quantified based on the extent of water penetration. The results of our calculation suggest that in these applications it may be pcesible to fit or model the protein density in the region of the boundary with solvent based on aqueous simulations to give a quantitative, smooth transition from protein to solvent. This is a very different picture of the surface of a protein than that given by the a-sible surface area of the crystallographic co. ordinates. Averaging over the fluctuations rather than giving them explicit consideration leads to a view of the protein-solvent interface as being sharp and in contrast to the more realistic picture given here.
Acknowledgment. We thank the National Institutes of Health, the Robert A. Welch Foundation, and Alfred P. Sloan Foundation for partial support of this work.
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References and Notes
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Figure 2. Same time average as in Figure 1 for a different c r w section of the protein. The solid contours represent the water density, as in Figure 1, and the dot contours depict the extent of protein density. The outer protein contour is the 5% density surface, and its placement with respect to the 5% water contour (no. 1) shows the extent of the inhomogeneous interpretation region at the interface with water.
For many crystallographic structure determinations, solvent flattening p d u r e s are used to avoid the problems associated with solvent density refinement.' In the area of crystallographic refinements that include water, density maps can be constructed to higbUght the water density.I8 In such calculations, subtracting the protein density as a stepfunction can lead to Fourier noise problems. It is also customary to use smoothing parameters that excccd typical DebytWaller factors, but justifications have not been made explicitly phy~ical.'~ Poisson-Boltzmann calculations of the electrostatic potential of a protein, useful for interpreting a number of phenomena including titration curves and solvation energies, have often used a sharp boundary to separate the protein dielectric from that of the aqueous solvent.20 Recent calculations have demonstrated improved numerical stability of the calculated solvation thermodynamics by using a slight tapering of the boundary.2' This
(1) Hendrickmn, W. A.; Konnert, J. H. In Computing in Crystallography; Diamond, R., Ed.; Indian Academy of Science: Bangalore, 1980; pp 1-25. Academic Ress: Blundell, T. L.; Johnson, L. N. Protein Crystallography; . . . New York, 1976. (2) Otting, G.; Liepinsh, E.; Wtkhrich, K. Science 1991, 254, 974-980. Bryant. R. G. Stud. Phvs. Theor. Chem. 1988. 38.683-705. *(3) Teeter, M. Ado. Biophys. Biochem. 1991, 20, 577. Teeter, M.; Ma, X.Q.;Rao, U.; Whitlow, M. Proteins 1990, 8, 118-132. (4) Parak, F. Comments Mol. Cell. Biophys. 1907, 4, 265-280. (5) McCammon J. A.; Harvey, S . C. Dynamics of Proteins and Nucleic Acids; Cambridge University Press: New York,2,3. Brooks,C. L.; Karplus, M.;Pettitt, B. M. Advances in Chemical Physics; John Wiley & Sons: New York, 1988; Vol. 71. ( 6 ) Levitt, M.; Sharon, R. Proc. Natl. Acad. Sci. U.S.A. 1988,85,7557. (7) Lee, E.;Richards, F. M. J. Mol. Biol. 1971, 55, 379-400. (8) Protein Data Bank, Chemistry Department, Brookhaven National Laboratory. (9) Richards, F . M. Carlsberg Res. Commun. 1979, 44, 47-63. .(10) Takano, T. J . Mol. Biol. 1977, 110, 537-568. (1 1) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W.F.; Hermans, J. In Intermolecular Forces; Pullmann, B., Ed.; Reidel: Dordreclht, 1981. (12) Weiner, S.J.; Kdlman, P. A,; Nguyen, D. T.; Case, D. A. J. Compur. Chem. 1986, 7 , 230-252. (13) Findscn, L. A,; Subramaniam, S.;Lounnas, V.;Pettitt, B. M. Molecular Dynamics Simulation of Metmyoglobin in Aqueous Solution. In Principles of Molecular Recognition; Buckingham, A. D., Ed.; Chapman and Hall: London, 1991. (14) Case, D.; Karplus, M. J. Mol. Biol. 1979, 132, 34. (15) Pettitt, B. M.; Karplus, M. Chem. Phys. Lett. 1985, 121, 194-201. Brooks,C. L.; Karplus, M. Methods Enzymol. 1986, 127, 369-400. (16) Friedman, H. L. A Course in Statistical Mechanics; Prcntice-Hall: Englewood Cliffs, NJ, 1985; pp 77-82.
J. Phys. Chem. 1992,96,7 159-7 161 (17) Badger, J.;Caspar, D. L. D. Proc. Narl. Acad. Sci. U S A . 1991,88, 622. (18) Cheng, X.; Schoenborn, B. P. Acta Crysraflogr. 1990, 816, 195. (19) Blake, C. C. F.; hlfd,W. C. A.; Artymiuk P. J. J. Mol. Biol. 1983, 167, 693.
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(20)Gilson, P.J.; Honig, B. Proteins 1!M, 4, 7-18. Tanford, C.; Kirkwood, J. G. J . Am. Chem. Soc. 1957,79, 5333-5339. (21) Davis, M. E.; McCammon, J. A. J. Compur. Chem. 1991, 12, 909-912. (22)Hagler, A.; Moult, J. Nature 1978, 272, 222.
Isomers and ''C Hyperfine Structures of MetaEEncapsulated Fullerenes M@Ca2(M = Sc, Y, and La) Shinzo S t u d , * Satoghi Kawata, Haruo Shiromaru, Kotaro Yamauchi, Koichi Kikuchi, T a t " Kat02 and Yohji Achiba* Department of Chemistry, Tokyo Metropolitan University, Hachioji, Tokyo 192-03, Japan, and Institute for Molecular Science, Myodaiji, Okazaki 444, Japan (Received: June 1, 1992; In Final Form: July 20, 1992)
The formation of two isomers of C& with a metal inside, M a c E 2(M = Sc,Y,and La) was fmt identified by ESR spectroscopy. The production ratio of two isomers found for M a c E 2suggests that the cage structure of the isomers of M a c E 2is closely associated with those of the empty Cs2. However, contrary to the existence of four or more distinct isomers of empty Cs2, the observation of only two metallofullerene isomers strongly suggests that a metal is selectively encapsulated in the CE2 cages with particular structures. An endohedral form of metal-containing CE2fullerene was established by observation of well-resolved ESR spectra of hyperfine coupling to I3C in natural abundance on the carbon cages.
Introduction Recent discovery of macroscopic preparations of metal-encapsulated fullerene M@Cs2(M = Sc, Y, and La) enabled us to study the nature of such an exotic molecular form in detail. Among the many fascinating issues concerning the nature of these metal-fullerene complexes,1* a central question is the following: why is cs2 so special? M@Cs2has been indicated to be the only metallofullereneto be extracted in solution except for the case of La2Cw2 Mass spectra of Sc-containingcrude extract indicated the existence of diatomic species Sc2C2,(2n = 80, 82,84, ...).s,6 In this sense, structural information on these endohedral complexes must be unambiguously important to understand the peculiar properties of these fullerenes. On the other hand, the cage structures of the empty Cs2have recently been successfully determined by Kikuchi et al., using 13C NMR in solution for the chromatographically separated C8z.7 According to their results, the major isomer of Cs2 has a C, symmetry, and at least three other minor isomers with C,, C,,, and C2(or C,) symmetries coexist. Therefore, it is interesting to investigate whether or not isomers exist even for the fullerene Cs2 with a metal inside. In the case of M a c s 2 (M = Sc, Y, and La), if odd numbers of electrm transfer from the metal embedded inside to the carbon cage outside, the cage would have an electron spin and show ESR signals. Furthermore, if there coexists isomers of metallofullerenes, each isomer would have a different g value as well as a different hyperfine coupling constant, which should be, in principle, distinguhhed from each other. The first expectation mentioned above was indeed the case in La@CS2for which the ESR spectrum caused by a half-spin on the carbon cage was actually observed by Johnson et ala3for the first time. For La@Cs2,they have shown eight equally spaced lines with an equal intensity centered at g = 2.0010. The eight ESR lines were well interpreted in terms of a hyperfine structure due to the '39Lanuclei with I = '/> They Collcluded that the ekctronicstructure Of La@%2 is well dacribed by La3+@Csf. Recently, Weaver et a1.4 have observed the ESR spectrum of Y@CS2,consisting of an I = l / 2 system. Shinohara et al.s and Yannoni et a1.6 more recently reported the ESR spectrum of the Sc,-Cs2 system (x = 1 and 3). To whom corre6pondence should be a d d r d . tInstitute for Molecular Science.
In this report, it is shown that a metal gets into two different CE2carbon cages with particular structures. The systematic measurements of ESR spectra for three metal-encapsulated fullerenes indicate that two isomers of M a c s 2 are commonly formed with almost the same fraction ratio. The possible cage candidates for these metalofullerenes are discussed by comparison with the results recently obtained from 13CNMR measurements on the empty Cg2fullerene. Exper&nenWSection The sample was prepared by a method essentially the same as those described already.'* Briefly, a mixture of graphite powder and metal oxide (MZOJ, to approximately 1 metal atom per 130 carbon atoms, was mixed with graphite cement (551-R, Aremco Products Inc.) to an approximately 1:3 volume ratio and pressed into a rod. After curing at 200 OC overnight, the rod was heated to 1200 OC for 10 h. The soot was produced by dc arc discharge of the rod under about 200-Torr He atmosphere. The soot was collected and extracted by CSPs Laser desorption timesf-flight mass spectra of crude extracts, taken by a negativeion mode, have shown that the fulkrene species associated with a single metal was YcS2 LacS2, and ScCs2.From the mass spectra,it was also found that there were no mass peaks due to impurity metals whose nuclear magnetic moments are accidentally the same as La, Y, or Sc. Therefore, it was confvmed that the species giving the ESR signals in the present work was due to MC82 (M = Sc, Y, and La), as was also indicated by Chai et a1.l and by Alvarez et aL2 and Johnson et aL3for h@c82, by Weaver et a1.4 for Y@Cg2,and by Shinohara et alq5and Yannoni et a1.6 for Sc@CE2. After each crude extract was dissolved into toluene or CS2and degassed, ESR measurements were performed using a conventional X-band ESR spectrometer (JEOL RE-3X).
Results and Discussion The X-band ESR spectra of the metallofullerenes MCS2(M = Sc, Y, La) in solution at room temperatureare shown in Figure 1, a,b, and c, respectively. The main features are essentially the same as those already reported for La@C82,3Y@Cs2,4and except the fact that there appears small, but wellresolved, additional lines for the present case. Therefore,we mostly focus on these small ESR lines in the present report.
0022-3654/92/2096-7 159$03.00/0 Q 1992 American Chemical Society
