Pathways to a protein folding intermediate observed in a 1-microsecond simulation in aqueous solution

Science

Volumn 282, Issue 5389, 1998, Pages 740-744

  Duan, Yong ^a   Kollman, Peter A ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

VILLIN; WATER;

AQUEOUS SOLUTION; ARTICLE; CHRONOLOGY; COMPUTER SIMULATION; CONFORMATIONAL TRANSITION; HYDROPHOBICITY; MOLECULAR DYNAMICS; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN FOLDING;

CARRIER PROTEINS; COMPUTER SIMULATION; MATHEMATICAL COMPUTING; MICROFILAMENT PROTEINS; MODELS, MOLECULAR; NEUROFILAMENT PROTEINS; NUCLEAR MAGNETIC RESONANCE, BIOMOLECULAR; PEPTIDE FRAGMENTS; PROTEIN CONFORMATION; PROTEIN FOLDING; PROTEIN STRUCTURE, SECONDARY; PROTEIN STRUCTURE, TERTIARY; THERMODYNAMICS;

EID: 0032561237     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5389.740     Document Type: Article

References (55)

1. R. N. Sifers, Nature Struct. Biol. 2, 355 (1995); S. B. Prusiner, Science 278, 245 (1997).
2. R. N. Sifers, Nature Struct. Biol. 2, 355 (1995); S. B. Prusiner, Science 278, 245 (1997).
3. Reviewed in K. A. Dill and H. S. Chan, Nature Struct. Biol. 4, 10 (1997); C. M. Dobson and O. B. Ptitsyn, Curr. Opin. Struct. Biol. 7, 1 (1997); E. Shakhnovich and A. R. Fersht, ibid. 8, 65 (1998).
4. Reviewed in K. A. Dill and H. S. Chan, Nature Struct. Biol. 4, 10 (1997); C. M. Dobson and O. B. Ptitsyn, Curr. Opin. Struct. Biol. 7, 1 (1997); E. Shakhnovich and A. R. Fersht, ibid. 8, 65 (1998).
5. Reviewed in K. A. Dill and H. S. Chan, Nature Struct. Biol. 4, 10 (1997); C. M. Dobson and O. B. Ptitsyn, Curr. Opin. Struct. Biol. 7, 1 (1997); E. Shakhnovich and A. R. Fersht, ibid. 8, 65 (1998).
6. J. Skolnick and A. Kolinski, Science 250, 1121 (1990); A. Sali, E. Shakhnovich, M. Karplus, Nature 369, 248 (1994); K. A. Dill et al., Protein Sci. 4, 561 (1995).
7. J. Skolnick and A. Kolinski, Science 250, 1121 (1990); A. Sali, E. Shakhnovich, M. Karplus, Nature 369, 248 (1994); K. A. Dill et al., Protein Sci. 4, 561 (1995).
8. J. Skolnick and A. Kolinski, Science 250, 1121 (1990); A. Sali, E. Shakhnovich, M. Karplus, Nature 369, 248 (1994); K. A. Dill et al., Protein Sci. 4, 561 (1995).
9. C. A. Schiffer, V. Dötsch, K. Wüthrich, W. F. van Gunsteren. Biochemistry 34, 15057 (1995).
10. T. Lazaridis and M. Karplus, Science 278, 1928 (1997).
11. T. Fox and P. A. Kollman, Proteins 25, 315 (1996); P. A. Kollman, Acc. Chem. Res. 29, 461 (1996).
12. T. Fox and P. A. Kollman, Proteins 25, 315 (1996); P. A. Kollman, Acc. Chem. Res. 29, 461 (1996).
13. The longest single MD trajectories of proteins with explicit water have been 5.4 ns [A. Li and V. Daggett, Protein Eng. 8, 1117 (1995)].
14. V. Daggett and M. Levitt, Proc. Natl. Acad. Sci. U.S.A. 89, 5142 (1992); J. Tirado-Rives and W. L. Jorgensen, Biochemistry 32, 4175 (1993); V. Daggett and M. Levitt, Curr. Opin. Struct. Biol. 4, 291 (1994).
15. V. Daggett and M. Levitt, Proc. Natl. Acad. Sci. U.S.A. 89, 5142 (1992); J. Tirado-Rives and W. L. Jorgensen, Biochemistry 32, 4175 (1993); V. Daggett and M. Levitt, Curr. Opin. Struct. Biol. 4, 291 (1994).
16. V. Daggett and M. Levitt, Proc. Natl. Acad. Sci. U.S.A. 89, 5142 (1992); J. Tirado-Rives and W. L. Jorgensen, Biochemistry 32, 4175 (1993); V. Daggett and M. Levitt, Curr. Opin. Struct. Biol. 4, 291 (1994).
17. Brooks and co-workers have attempted to reconstruct the folding free-energy landscape [E. M. Boczko and C. L. Brooks III, Science 269, 393 (1995); F. B. Sheinerman and C. L. Brooks III, Mol. Biol. 278, 439 (1998)] from the restrained unfolding simulations using the WHAM method [S. Kumar, D. Bouzida, R. H. Swendsen, P. A. Kollman, J. M. Rosenberg, J. Comp. Chem. 13, 1011 (1992); E. M. Boczko and C. L. Brooks III, Phys. Chem. 97, 4509 (1993)].
18. Brooks and co-workers have attempted to reconstruct the folding free-energy landscape [E. M. Boczko and C. L. Brooks III, Science 269, 393 (1995); F. B. Sheinerman and C. L. Brooks III, Mol. Biol. 278, 439 (1998)] from the restrained unfolding simulations using the WHAM method [S. Kumar, D. Bouzida, R. H. Swendsen, P. A. Kollman, J. M. Rosenberg, J. Comp. Chem. 13, 1011 (1992); E. M. Boczko and C. L. Brooks III, Phys. Chem. 97, 4509 (1993)].
19. Brooks and co-workers have attempted to reconstruct the folding free-energy landscape [E. M. Boczko and C. L. Brooks III, Science 269, 393 (1995); F. B. Sheinerman and C. L. Brooks III, Mol. Biol. 278, 439 (1998)] from the restrained unfolding simulations using the WHAM method [S. Kumar, D. Bouzida, R. H. Swendsen, P. A. Kollman, J. M. Rosenberg, J. Comp. Chem. 13, 1011 (1992); E. M. Boczko and C. L. Brooks III, Phys. Chem. 97, 4509 (1993)].
20. Brooks and co-workers have attempted to reconstruct the folding free-energy landscape [E. M. Boczko and C. L. Brooks III, Science 269, 393 (1995); F. B. Sheinerman and C. L. Brooks III, Mol. Biol. 278, 439 (1998)] from the restrained unfolding simulations using the WHAM method [S. Kumar, D. Bouzida, R. H. Swendsen, P. A. Kollman, J. M. Rosenberg, J. Comp. Chem. 13, 1011 (1992); E. M. Boczko and C. L. Brooks III, Phys. Chem. 97, 4509 (1993)].
21. E. Demchuk, D. Bashford, D. A. Case, Fold. Des. 2, 35 (1997); X. Daura, B. Jaun, D. Seebach, W. F. van Gunsteren, A. E. Mark, J. Mol. Biol. 280, 925 (1998).
22. E. Demchuk, D. Bashford, D. A. Case, Fold. Des. 2, 35 (1997); X. Daura, B. Jaun, D. Seebach, W. F. van Gunsteren, A. E. Mark, J. Mol. Biol. 280, 925 (1998).
23. F. B. Sheinerman and C. L. Brooks, Proc. Natl. Acad. Sci. U.S.A. 95, 1562 (1998).
24. E. I. Shakhnovich, Curr. Opin. Struct. Biol. 7, 29 (1997).
25. Y. Duan, L. Wang, P. A. Kollman, Proc. Natl. Acad. Sci. U.S.A. 95, 9897 (1998).
26. C. J. McKnight, D. S. Doering, P. T. Matsudarira, P. S. Kim, J. Mol. Biol. 260, 126 (1996).
27. C. J. McKnight, P. T. Matsudaira, P. S. Kim, Nature Struct. Biol. 4, 180 (1997).
28. The force field [W. D. Cornell et al., J. Am. Chem. Soc. 117, 5179 (1995)] of Cornell et al. was used with full representation of solvent with the TIP3P water model [W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, J. Chem. Phys. 79, 926 (1983)]. Periodic boundary conditions were imposed by a nearest image convention in a truncated octahedron box. An 8 Å residue-based cutoff was applied to the long-range nonbonded protein-water and water-water interactions (both electrostatic and van der Waals). The intramolecular nonbonded interactions of protein were calculated without truncation. When applicable, temperature and pressure controls were imposed through use of Berendsen's algorithms [H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Comp. Phys. 81, 3684 (1984)]. The solute and solvent were separately coupled to a temperature bath with coupling constants of 0.1 ps. The pressure coupling constant was 20 ps. The trajectories were produced by numerical integration with the Verlet-leapfrog algorithm [L. Verlet, Phys. Rev. 159, 98 (1967); C. L. Brooks III, M. Karplus, B. M. Pettitt, Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Wiley, New York, 1989)] by using a 2-fs time step. Bond constraints were imposed on all bonds involving hydrogen atoms with SHAKE [J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comp. Phys. 23, 327 (1977)] and SETTLE [S. Miyamoto and P. A. Kollman, J. Comp. Chem. 13, 952 (1992)].
29. The force field [W. D. Cornell et al., J. Am. Chem. Soc. 117, 5179 (1995)] of Cornell et al. was used with full representation of solvent with the TIP3P water model [W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, J. Chem. Phys. 79, 926 (1983)]. Periodic boundary conditions were imposed by a nearest image convention in a truncated octahedron box. An 8 Å residue-based cutoff was applied to the long-range nonbonded protein-water and water-water interactions (both electrostatic and van der Waals). The intramolecular nonbonded interactions of protein were calculated without truncation. When applicable, temperature and pressure controls were imposed through use of Berendsen's algorithms [H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Comp. Phys. 81, 3684 (1984)]. The solute and solvent were separately coupled to a temperature bath with coupling constants of 0.1 ps. The pressure coupling constant was 20 ps. The trajectories were produced by numerical integration with the Verlet-leapfrog algorithm [L. Verlet, Phys. Rev. 159, 98 (1967); C. L. Brooks III, M. Karplus, B. M. Pettitt, Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Wiley, New York, 1989)] by using a 2-fs time step. Bond constraints were imposed on all bonds involving hydrogen atoms with SHAKE [J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comp. Phys. 23, 327 (1977)] and SETTLE [S. Miyamoto and P. A. Kollman, J. Comp. Chem. 13, 952 (1992)].
30. The force field [W. D. Cornell et al., J. Am. Chem. Soc. 117, 5179 (1995)] of Cornell et al. was used with full representation of solvent with the TIP3P water model [W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, J. Chem. Phys. 79, 926 (1983)]. Periodic boundary conditions were imposed by a nearest image convention in a truncated octahedron box. An 8 Å residue-based cutoff was applied to the long-range nonbonded protein-water and water-water interactions (both electrostatic and van der Waals). The intramolecular nonbonded interactions of protein were calculated without truncation. When applicable, temperature and pressure controls were imposed through use of Berendsen's algorithms [H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Comp. Phys. 81, 3684 (1984)]. The solute and solvent were separately coupled to a temperature bath with coupling constants of 0.1 ps. The pressure coupling constant was 20 ps. The trajectories were produced by numerical integration with the Verlet-leapfrog algorithm [L. Verlet, Phys. Rev. 159, 98 (1967); C. L. Brooks III, M. Karplus, B. M. Pettitt, Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Wiley, New York, 1989)] by using a 2-fs time step. Bond constraints were imposed on all bonds involving hydrogen atoms with SHAKE [J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comp. Phys. 23, 327 (1977)] and SETTLE [S. Miyamoto and P. A. Kollman, J. Comp. Chem. 13, 952 (1992)].
31. The force field [W. D. Cornell et al., J. Am. Chem. Soc. 117, 5179 (1995)] of Cornell et al. was used with full representation of solvent with the TIP3P water model [W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, J. Chem. Phys. 79, 926 (1983)]. Periodic boundary conditions were imposed by a nearest image convention in a truncated octahedron box. An 8 Å residue-based cutoff was applied to the long-range nonbonded protein-water and water-water interactions (both electrostatic and van der Waals). The intramolecular nonbonded interactions of protein were calculated without truncation. When applicable, temperature and pressure controls were imposed through use of Berendsen's algorithms [H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Comp. Phys. 81, 3684 (1984)]. The solute and solvent were separately coupled to a temperature bath with coupling constants of 0.1 ps. The pressure coupling constant was 20 ps. The trajectories were produced by numerical integration with the Verlet-leapfrog algorithm [L. Verlet, Phys. Rev. 159, 98 (1967); C. L. Brooks III, M. Karplus, B. M. Pettitt, Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Wiley, New York, 1989)] by using a 2-fs time step. Bond constraints were imposed on all bonds involving hydrogen atoms with SHAKE [J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comp. Phys. 23, 327 (1977)] and SETTLE [S. Miyamoto and P. A. Kollman, J. Comp. Chem. 13, 952 (1992)].
32. The force field [W. D. Cornell et al., J. Am. Chem. Soc. 117, 5179 (1995)] of Cornell et al. was used with full representation of solvent with the TIP3P water model [W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, J. Chem. Phys. 79, 926 (1983)]. Periodic boundary conditions were imposed by a nearest image convention in a truncated octahedron box. An 8 Å residue-based cutoff was applied to the long-range nonbonded protein-water and water-water interactions (both electrostatic and van der Waals). The intramolecular nonbonded interactions of protein were calculated without truncation. When applicable, temperature and pressure controls were imposed through use of Berendsen's algorithms [H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Comp. Phys. 81, 3684 (1984)]. The solute and solvent were separately coupled to a temperature bath with coupling constants of 0.1 ps. The pressure coupling constant was 20 ps. The trajectories were produced by numerical integration with the Verlet-leapfrog algorithm [L. Verlet, Phys. Rev. 159, 98 (1967); C. L. Brooks III, M. Karplus, B. M. Pettitt, Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Wiley, New York, 1989)] by using a 2-fs time step. Bond constraints were imposed on all bonds involving hydrogen atoms with SHAKE [J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comp. Phys. 23, 327 (1977)] and SETTLE [S. Miyamoto and P. A. Kollman, J. Comp. Chem. 13, 952 (1992)].
33. The force field [W. D. Cornell et al., J. Am. Chem. Soc. 117, 5179 (1995)] of Cornell et al. was used with full representation of solvent with the TIP3P water model [W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, J. Chem. Phys. 79, 926 (1983)]. Periodic boundary conditions were imposed by a nearest image convention in a truncated octahedron box. An 8 Å residue-based cutoff was applied to the long-range nonbonded protein-water and water-water interactions (both electrostatic and van der Waals). The intramolecular nonbonded interactions of protein were calculated without truncation. When applicable, temperature and pressure controls were imposed through use of Berendsen's algorithms [H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Comp. Phys. 81, 3684 (1984)]. The solute and solvent were separately coupled to a temperature bath with coupling constants of 0.1 ps. The pressure coupling constant was 20 ps. The trajectories were produced by numerical integration with the Verlet-leapfrog algorithm [L. Verlet, Phys. Rev. 159, 98 (1967); C. L. Brooks III, M. Karplus, B. M. Pettitt, Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Wiley, New York, 1989)] by using a 2-fs time step. Bond constraints were imposed on all bonds involving hydrogen atoms with SHAKE [J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comp. Phys. 23, 327 (1977)] and SETTLE [S. Miyamoto and P. A. Kollman, J. Comp. Chem. 13, 952 (1992)].
34. The force field [W. D. Cornell et al., J. Am. Chem. Soc. 117, 5179 (1995)] of Cornell et al. was used with full representation of solvent with the TIP3P water model [W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, J. Chem. Phys. 79, 926 (1983)]. Periodic boundary conditions were imposed by a nearest image convention in a truncated octahedron box. An 8 Å residue-based cutoff was applied to the long-range nonbonded protein-water and water-water interactions (both electrostatic and van der Waals). The intramolecular nonbonded interactions of protein were calculated without truncation. When applicable, temperature and pressure controls were imposed through use of Berendsen's algorithms [H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Comp. Phys. 81, 3684 (1984)]. The solute and solvent were separately coupled to a temperature bath with coupling constants of 0.1 ps. The pressure coupling constant was 20 ps. The trajectories were produced by numerical integration with the Verlet-leapfrog algorithm [L. Verlet, Phys. Rev. 159, 98 (1967); C. L. Brooks III, M. Karplus, B. M. Pettitt, Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Wiley, New York, 1989)] by using a 2-fs time step. Bond constraints were imposed on all bonds involving hydrogen atoms with SHAKE [J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comp. Phys. 23, 327 (1977)] and SETTLE [S. Miyamoto and P. A. Kollman, J. Comp. Chem. 13, 952 (1992)].
35. 3 within 10 ps, and remained so for the remainder of the 1-ns solvent equilibration trajectory. The simulation was then conducted for 1.0 μ.s (0.5 billion integration steps). Temperature and pressure were controlled at physiological conditions (that is, 300 K and 1 atm) by the methods described above. Both temperature and density stabilized within 10 ps. The trajectory was saved at 20-ps intervals for the analysis. The entire simulation took ∼2 months' CPU time on a 256-CPU Cray T3D and an equal amount of CPU time on a 256-CPU Cray T3E-600.
36. 3 within 10 ps, and remained so for the remainder of the 1-ns solvent equilibration trajectory. The simulation was then conducted for 1.0 μ.s (0.5 billion integration steps). Temperature and pressure were controlled at physiological conditions (that is, 300 K and 1 atm) by the methods described above. Both temperature and density stabilized within 10 ps. The trajectory was saved at 20-ps intervals for the analysis. The entire simulation took ∼2 months' CPU time on a 256-CPU Cray T3D and an equal amount of CPU time on a 256-CPU Cray T3E-600.
37. The use of nonbonded cutoffs in the simulation, as performed here, is an inherent source of "noise" in the simulation trajectory. Such noise is not as deleterious in small proteins with limited formal charges as it is in, for example, RNA and DNA [T. E. Cheatham III, J. L Miller, T. Fox, T. A. Darden, P. A. Kollman, J. Am. Chem. Soc. 117, 4193 (1995)], as demonstrated by the stability of the 100-ns simulation at 300 K started from the native NMR structure. But accurate inclusion of long-range electrostatic effects [U. Essmann et al., J. Chem. Phys. 103, 8577 (1995)] does provide an additional challenge for achieving high parallelism in the MD code. Work is in progress to include long-range electrostatic effects while still achieving a level of parallelism and speed comparable to that presented here.
38. The use of nonbonded cutoffs in the simulation, as performed here, is an inherent source of "noise" in the simulation trajectory. Such noise is not as deleterious in small proteins with limited formal charges as it is in, for example, RNA and DNA [T. E. Cheatham III, J. L Miller, T. Fox, T. A. Darden, P. A. Kollman, J. Am. Chem. Soc. 117, 4193 (1995)], as demonstrated by the stability of the 100-ns simulation at 300 K started from the native NMR structure. But accurate inclusion of long-range electrostatic effects [U. Essmann et al., J. Chem. Phys. 103, 8577 (1995)] does provide an additional challenge for achieving high parallelism in the MD code. Work is in progress to include long-range electrostatic effects while still achieving a level of parallelism and speed comparable to that presented here.
39. O. B. Ptitsyn, Curr. Opin. Struct. Biol. 5, 74 (1995).
40. R. M. Ballew, J. Sabelko, M. Gruebele, Proc. Natl. Acad. Sci. U.S.A. 93, 5759 (1996).
41. R. E. Burton, C. S. Huang, M. A. Daugherty, T. L. Calderone, T. G. Oas, Nature Struct. Biol. 4, 305 (1997).
42. S. J. Hagen, J. Hofrichter, A. Szabo, W. A. Eaton, Proc. Natl. Acad. Sci. U.S.A. 93, 11615 (1996); W. A. Eaton, V. Muñoz, P. A. Thompson, C. K. Chan, J. Hofrichter, Curr. Opin. Struct. Biol. 7, 10 (1997).
43. S. J. Hagen, J. Hofrichter, A. Szabo, W. A. Eaton, Proc. Natl. Acad. Sci. U.S.A. 93, 11615 (1996); W. A. Eaton, V. Muñoz, P. A. Thompson, C. K. Chan, J. Hofrichter, Curr. Opin. Struct. Biol. 7, 10 (1997).
44. K. W. Plaxco, personal communication (1998).
45. R. Gilmanshin, S. Williams, R. H. Callender, W. H. Woodruff, R. B. Dyer, Proc. Natl. Acad. Sci. U.S.A. 94, 3709 (1997).
46. S. Williams et al., Biochemistry 35, 691 (1996); P. A. Thompson, W. A. Eaton, J. Hofrichter, ibid. 36, 9200 (1997).
47. S. Williams et al., Biochemistry 35, 691 (1996); P. A. Thompson, W. A. Eaton, J. Hofrichter, ibid. 36, 9200 (1997).
48. D. O. V. Alonso and V. Daggett, J. Mol. Biol. 247, 501 (1995); Protein Sci. 7, 860 (1998).
49. D. O. V. Alonso and V. Daggett, J. Mol. Biol. 247, 501 (1995); Protein Sci. 7, 860 (1998).
50. C. B. Anfinsen, Science 181, 223 (1973).
51. A total of 50,000 sets of coordinates were accumulated every 20 ps. They were clustered by comparison with the average coordinate of existing clusters using a 3.0 Å main-chain rmsd cutoff, similar to the method described by Karpen et al. [M. E. Karpen, D. J. Tobias, C. L. Brooks III, Biochemistry 32, 412 (1993)]. Those that are within 3.0 Å main-chain rmsd from the average coordinates of the cluster are assigned to the cluster. A total of 98 clusters were produced. Thirty clusters were highly populated with ∼500 or more coordinate sets and 13 clusters had more than 1000 sets of coordinates.
52. K. W. Plaxco, K. T. Simons, D. Baker, J. Mol. Biol. 277, 985 (1998).
53. Y. Duan and P. A. Kollman, unpublished data.
54. D. Eisenberg and A. D. McLachlan, Nature 319, 199 (1986).
55. Supercomputing time was provided by Cray Research, a subsidiary of Silicon Graphics, Inc. (SGI), and by the Pittsburgh Supercomputing Center (PSC). We are grateful to R. Roskies and M. Levine (PSC), J. Carpenter and H. Pritchard (SGI), and J. Wendoloski (AMGEN) for their support. We thank K. Dill, D. Agard, I. Kuntz, J. Pitera, and T. Cheatham for critical reading of the manuscript; L. Wang, C. Simmerling, M. Crowley, J. Wang, and W. Wang for stimulating discussions; and L. Chiche for the solvation free-energy calculation program. Graphics were provided by Computer Graphics Lab of the University of California, San Francisco (T. Ferrin, Principal Investigator, grant RR-1081). This work was supported in part by NIH grant GM-29072, by a University of California Biotechnology Star grant, and by AMGEN (P.A.K.).