Mutational analysis of the active site flap (20s loop) of mandelate racemase

Biochemistry

Volumn 47, Issue 2, 2008, Pages 566-578

  Bourque, Jennifer R ^a   Bearne, Stephen L ^a  

^a DALHOUSIE UNIVERSITY   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

MUTATIONAL ANALYSIS; STERIC SUBSTITUTIONS;

CATALYST ACTIVITY; HYDROPHOBICITY; LIGANDS; MAGNESIUM; PROTON TRANSFER; VAN DER WAALS FORCES;

ENANTIOMERS;

2 NAPHTHYLGLYCOLATE; ALANINE; GLYCOLIC ACID DERIVATIVE; MAGNESIUM; MANDELATE RACEMASE; MANDELIC ACID; NAPHTHYL GROUP; PHENYL GROUP; SOLVENT; UNCLASSIFIED DRUG;

ARTICLE; BETA SHEET; ENANTIOMER; ENZYME ACTIVE SITE; ENZYME MECHANISM; ENZYME SPECIFICITY; HYDROPHOBICITY; MOLECULAR INTERACTION; MOLECULAR SIZE; MUTATION; NONHUMAN; PRIORITY JOURNAL; PROTON TRANSPORT; PSEUDOMONAS PUTIDA; STEREOSPECIFICITY; WILD TYPE;

BINDING SITES; CIRCULAR DICHROISM; DNA MUTATIONAL ANALYSIS; GLYCOLATES; ISOMERISM; KINETICS; MAGNESIUM; MANDELIC ACIDS; MODELS, MOLECULAR; MUTANT PROTEINS; MUTATION; PSEUDOMONAS PUTIDA; RACEMASES AND EPIMERASES; SUBSTRATE SPECIFICITY;

PSEUDOMONAS PUTIDA;

EID: 38849175459     PISSN: 00062960     EISSN: None     Source Type: Journal    
DOI: 10.1021/bi7015525     Document Type: Article

References (99)

1. Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, S., and Petsko, G. A. (1994) The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: Mechanistic and crystallographic evidence for stereospecific alkylation by (R)-α-phenylglycidate, Biochemistry 33, 635-643.
2. Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R. S., Kenyon, G. L., and Gerlt, J. A. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-Å resolution: Identification of the active site and possible catalytic residues, Biochemistry 30, 9264-9273.
3. Kallarakal, A. T., Mitra, B., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., and Kenyon, G. L. (1995) Mechanism of the reaction catalyzed by mandelate racemase: Structure and mechanistic properties of the K166R mutant, Biochemistry 34, 2788-2797.
4. Leszczynski, J. F., and Rose, G. D. (1986) Loops in globular proteins: A novel category of secondary structure, Science 234, 849-855.
5. Knowles, J. R. (1991) Enzyme catalysis: Not different, just better, Nature 350, 121-124.
6. Kempner, E. S. (1993) Movable lobes and flexible loops in proteins. Structural deformations that control biochemical activity, FEBS Lett. 326, 4-10.
7. Gerstein, M., Lesk, A. M., and Chothia, C. (1994) Structural mechanisms for domain movements in proteins, Biochemistry 33, 6739-6749.
8. Fetrow, J. S. (1995) Omega loops: Nonregular secondary structures significant in protein function and stability, FASEB J. 9, 708-717.
9. Wang, G. P., Cahill, S. M., Liu, X., Girvin, M. E., and Grubmeyer, C. (1999) Motional dynamics of the catalytic loop in OMP synthase, Biochemistry 38, 284-295.
10. Wierenga, R. K. (2001) The TIM-barrel fold: A versatile framework for efficient enzymes, FEBS Lett. 492, 193-198.
11. Berlow, R. B., Igumenova, T. I., and Loria, J. P. (2007) Value of a hydrogen bond in triosephosphate isomerase loop motion, Biochemistry 46, 6001-6010.
12. Kempf, J. G., Jung, J. Y., Ragain, C., Sampson, N. S., and Loria, J. P. (2007) Dynamic requirements for a functional protein hinge, J. Mol. Biol. 368, 131-149.
13. Casteleijn, M. G., Alahuhta, M., Groebel, K., El-Sayed, I., Augustyns, K., Lambeir, A. M., Neubauer, P., and Wierenga, R. K. (2006) Functional role of the conserved active site proline of triosephosphate isomerase, Biochemistry 45, 15483-15494.
14. Massi, F., Wang, C., and Palmer, A. G. (2006) Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase, Biochemistry 45, 10787-10794.
15. Eaazhisai, K., Balaram, H., Balaram, P., and Murthy, M. R. N. (2004) Structures of unliganded and inhibitor complexes of W168F, a loop6 hinge mutant of Plasmodium falciparum triosephosphate isomerase: Observation of an intermediate position of loop6, J. Mol. Biol. 343, 671-684.
16. Kursula, I., Salin, M., Sun, J., Norledge, B. V., Haapalainen, A. M., Sampson, N. S., and Wierenga, R. K. (2004) Understanding protein lids: Structural analysis of active hinge mutants in triosephosphate isomerase, Protein Eng., Des. Sel. 17, 375-382.
17. Xiang, J., Jung, J. Y., and Sampson, N. S. (2004) Entropy effects on protein hinges: The reaction catalyzed by triosephosphate isomerase, Biochemistry 43, 11436-11445.
18. Xiang, J., Sun, J., and Sampson, N. S. (2001) The importance of hinge sequence for loop function and catalytic activity in the reaction catalyzed by triosephosphate isomerase, J. Mol. Biol. 307, 1103-1112.
19. Rozovsky, S., Jogl, G., Tong, L., and McDermott, A. E. (2001) Solution-state NMR investigations of triosephosphate isomerase active site loop motion: Ligand release in relation to active site loop dynamics, J. Mol. Biol. 310, 271-280.
20. Rozovsky, S., and McDermott, A. E. (2001) The time scale of the catalytic loop motion in triosephosphate isomerase, J. Mol. Biol. 310, 259-270.
21. Gerlt, J. A. (1998) Enzyme-catalyzed proton transfer reactions to and from carbon, in Bioorganic Chemistry: Peptides and Proteins (Hecht, S. M., Ed.) pp 279-311, Oxford University Press, New York.
22. Babbitt, P. C., and Gerlt, J. A. (1997) Understanding enzyme superfamilies. Chemistry as the fundamental determinant in the evolution of new catalytic activities, J. Biol. Chem. 272, 30591-30594.
23. Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R., Barrett, W. C., Reed, G. H., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) The enolase superfamily: A general strategy for enzyme-catalyzed abstraction of the α-protons of carboxylic acids, Biochemistry 35, 16489-16501.
24. Gerlt, J. A., Kenyon, G. L., Kozarich, J. W., Neidhart, D. C., and Petsko, G. A. (1992) Mandelate racemase and class-related enzymes, Curr. Opin. Struct. Biol. 2, 736-742.
25. Gerlt, J. A., and Gassman, P. G. (1993) Understanding the rates of certain enzyme-catalyzed reactions: Proton abstraction from carbon acids, acyl-transfer reactions, and displacement reactions of phosphodiesters, Biochemistry 32, 11943-11952.
26. Gerlt, J. A., Babbitt, P. C., and Rayment, I. (2005) Divergent evolution in the enolase superfamily: The interplay of mechanism and specificity, Arch. Biochem. Biophys. 433, 59-70.
27. Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., and Kozarich, J. W. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism, Biochemistry 30, 9255-9263.
28. 8-barrels: In vitro enhancement of a "new" reaction in the enolase superfamily, Biochemistry 44, 11722-11729.
29. Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis, Biochemistry 44, 9013-9021.
30. Gerlt, J. A., and Babbitt, P. C. (2001) Divergent evolution of enzymatic function: Mechanistically diverse superfamilies and functionally distinct suprafamilies, Annu. Rev. Biochem. 70, 209-246.
31. Babbitt, P. C. (2003) Definitions of enzyme function for the structural genomics era, Curr. Opin. Chem. Biol. 7, 230-237.
32. Sampson, N. S., and Knowles, J. R. (1992) Segmental motion in catalysis: Investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase, Biochemistry 31, 8488-8494.
33. Sampson, N. S., and Knowles, J. R. (1992) Segmental movement: Definition of the structural requirements for loop closure in catalysis by triosephosphate isomerase, Biochemistry 31, 8482-8487.
34. Sun, J., and Sampson, N. S. (1998) Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase, Protein Sci. 7, 1495-1505.
35. Sun, J., and Sampson, N. S. (1999) Understanding protein lids: Kinetic analysis of active hinge mutants in triosephosphate isomerase, Biochemistry 38, 11474-11481.
36. Williams, J. C., and McDermott, A. E. (1995) Dynamics of the flexible loop of triosephosphate isomerase: The loop motion is not ligand gated, Biochemistry 34, 8309-8319.
37. Pompliano, D. L., Peyman, A., and Knowles, J. R. (1990) Stabilization of a reaction intermediate as a catalytic device: Definition of the functional role of the flexible loop in triosephosphate isomerase, Biochemistry 29, 3186-3194.
38. Amyes, T. L., and Richard, J. P. (2007) Enzymatic catalysis of proton transfer at carbon: Activation of triosephosphate isomerase by phosphite dianion, Biochemistry 46, 5841-5854.
39. Mitra, B., Kallarakal, A. T., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., and Kenyon, G. L. (1995) Mechanism of the reaction catalyzed by mandelate racemase: Importance of electrophilic catalysis by glutamic acid 317, Biochemistry 34, 2777-2787.
40. Schafer, S. L., Barrett, W. C., Kallarakal, A. T., Mitra, B., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., and Kenyon, G. L. (1996) Mechanism of the reaction catalyzed by mandelate racemase: Structure and mechanistic properties of the D270N mutant, Biochemistry 35, 5662-5669.
41. St. Maurice, M., and Bearne, S. L. (2000) Reaction intermediate analogues for mandelate racemase: Interaction between Asn 197 and the α-hydroxyl of the substrate promotes catalysis, Biochemistry 39, 13324-13335.
42. St. Maurice, M., and Bearne, S. L. (2004) Hydrophobic nature of the active site of mandelate racemase, Biochemistry 43, 2524-2532.
43. Bearne, S. L., St. Maurice, M., and Vaughan, M. D. (1999) An assay for mandelate racemase using high-performance liquid chromatography, Anal. Biochem. 269, 332-336.
44. Sharp, T. R., Hegeman, G. D., and Kenyon, G. L. (1979) A direct kinetic assay for mandelate racemase using circular dichroic measurements, Anal. Biochem. 94, 329-334.
45. Fee, J. A., Hegeman, G. D., and Kenyon, G. L. (1974) Mandelate racemase from Pseudomonas putida. Subunit composition and absolute divalent metal ion requirement, Biochemistry 13, 2528-2532.
46. Andrade, M. A., Chacon, P., Merelo, J. J., and Moran, F. (1993) Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network, Protein Eng. 6, 383-390.
47. Felfer, U., Goriup, M., Koegl, M. F., Wagner, U., Larissegger-Schnell, B., Faber, K., and Kroutil, W. (2005) The substrate spectrum of mandelate racemase: Minimum structural requirements for substrates and substrate model, Adv. Synth. Catal. 347, 951-961.
48. Larissegger-Schnell, B., Kroutil, W., and Faber, K. (2005) Chemoenzymatic synthesis of (R)- and (S)-2-hydroxy-4-phenylbutanoic acid via enantio-complementary deracemization of (±)-2-hydroxy-4-phenyl-3-butenoic acid using a racemase-lipase two-enzyme-system, Synlett 12, 1936-1938.
49. Strauss, U. T., and Faber, K. (1999) Deracemization of (±)-mandelic acid using a lipase-mandelate racemase two-enzyme system, Tetrahedron: Asymmetry 10, 4079-4081.
50. Felfer, U., Strauss, U. T., Kroutil, W., Fabian, W. M. F., and Faber, K. (2001) Substrate spectrum of mandelate racemase: Part 2. (Hetero)-aryl- substituted mandelate derivatives and modulation of activity, J. Mol. Catal. B: Enzym. 15, 213-222.
51. Kenyon, G. L., and Hegeman, G. D. (1970) Mandelic acid racemase from Pseudomonas putida. Evidence favoring a carbanion intermediate in the mechanism of action, Biochemistry 9, 4036-4043.
52. St. Maurice, M., and Bearne, S. L. (2002) Kinetics and thermodynamics of mandelate racemase catalysis, Biochemistry 41, 4048-4058.
53. Lin, D. T., Powers, V. M., Reynolds, L. J., Whitman, C. P., Kozarich, J. W., and Kenyon, G. L. (1988) Evidence for the generation of α-carboxy- α-hydroxy-p-xylylene from p-(bromomethyl)mandelate by mandelate racemase, J. Am. Chem. Soc. 110, 323-324.
54. Landro, J. A., Kenyon, G. L., and Kozarich, J. W. (1992) Mechanism-based inactivation of mandelate racemase by propargylglycolate, Bioorg. Med. Chem. Lett. 2, 1411-1418.
55. Li, R., Powers, V. M., Kozarich, J. W., and Kenyon, G. L. (1995) Racemization of vinylglycolate catalyzed by mandelate racemase, J. Org. Chem. 60, 3347-3351.
56. Fersht, A. (1999) Structure and Mechanism in Protein Science, W. H. Freeman and Co., New York.
57. Bordo, D., and Argos, P. (1991) Suggestions for "safe" residue substitutions in site-directed mutagenesis, J. Mol. Biol. 217, 721-729.
58. Chothia, C. (1975) Structural invariants in protein folding, Nature 254, 304-308.
59. Kraut, D. A., Carroll, K. S., and Herschlag, D. (2003) Challenges in enzyme mechanism and energetics, Annu. Rev. Biochem. 72, 517-571.
60. Mildvan, A. S. (2004) Inverse thinking about double mutants of enzymes, Biochemistry 43, 14517-14520.
61. Toth, G., and Borics, A. (2006) Flap opening mechanism of HIV-1 protease, J. Mol. Graphics Modell. 24, 465-474.
62. Kato, H., Tanaka, T., Yamaguchi, H., Hara, T., Nishioka, T., Katsube, Y., and Oda, J. (1994) Flexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase, Biochemistry 33, 4995-4999.
63. Larson, E. M., Larimer, F. W., and Hartman, F. C. (1995) Mechanistic insights provided by deletion of a flexible loop at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase, Biochemistry 34, 4531-4537.
64. Tanaka, T., Yamaguchi, H., Kato, H., Nishioka, T., Katsube, Y., and Oda, J. (1993) Flexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase, Biochemistry 32, 12398-12404.
65. 2+) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-Å resolution, Biochemistry 33, 9333-9342.
66. Brewer, J. M., Glover, C. V., Holland, M. J., and Lebioda, L. (1998) Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism, Biochim. Biophys. Acta 1383, 351-355.
67. Poyner, R. R., Larsen, T. M., Wong, S. W., and Reed, G. H. (2002) Functional and structural changes due to a serine to alanine mutation in the active-site flap of enolase, Arch. Biochem. Biophys. 401, 155-163.
68. Fisher, L. M., Albery, W. J., and Knowles, J. R. (1986) Energetics of proline racemase: Racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes, Biochemistry 25, 2529-2537.
69. Glavas, S., and Tanner, M. E. (2001) Active site residues of glutamate racemase, Biochemistry 40, 6199-6204.
70. Glavas, S., and Tanner, M. E. (1999) Catalytic acid/base residues of glutamate racemase, Biochemistry 38, 4106-4113.
71. Dodd, D., Reese, J. G., Louer, C. R., Ballard, J. D., Spies, M. A., and Blanke, S. R. (2007) Functional comparison of the two Bacillus anthracis glutamate racemases, J. Bacteriol. 189, 5265-5275.
72. Lundqvist, T., Fisher, S. L., Kern, G., Folmer, R. H., Xue, Y., Newton, D. T., Keating, T. A., Alm, R. A., and de Jonge, B. L. (2007) Exploitation of structural and regulatory diversity in glutamate racemases, Nature 447, 817-822.
73. May, M., Mehboob, S., Mulhearn, D. C., Wang, Z., Yu, H., Thatcher, G. R., Santarsiero, B. D., Johnson, M. E., and Mesecar, A. D. (2007) Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design, J. Mol. Biol. 371, 1219-1237.
74. Cleland, W. W. (1963) The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations, Biochim. Biophys. Acta 67, 104-137.
75. Host, G., Martensson, L. G., and Jonsson, B. H. (2006) Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, Biochim. Biophys. Acta 1764, 1601-1606.
76. 8-barrel protein, for modulation of the substrate specificity, Biochemistry 43, 7413-7420.
77. Suzuki, J., Sasaki, K., Sasao, Y., Hamu, A., Kawasaki, H., Nishiyama, M., Horinouchi, S., and Beppu, T. (1989) Alteration of catalytic properties of chymosin by site-directed mutagenesis, Protein Eng. 2, 563-569.
78. Shintani, T., Nomura, K., and Ichishima, E. (1997) Engineering of porcine pepsin. Alteration of S1 substrate specificity of pepsin to those of fungal aspartic proteinases by site-directed mutagenesis, J. Biol. Chem. 272, 18855-18861.
79. Okoniewska, M., Tanaka, T., and Yada, R. Y. (1999) The role of the flap residue, threonine 77, in the activation and catalytic activity of pepsin A, Protein Eng. 12, 55-61.
80. Wilks, H. M., Moreton, K. M., Halsall, D. J., Hart, K. W., Sessions, R. D., Clarke, A. R., and Holbrook, J. J. (1992) Design of a specific phenyllactate dehydrogenase by peptide loop exchange on the Bacillus stearothermophilus lactate dehydrogenase framework, Biochemistry 31, 7802-7806.
81. 8-barrels: Functional promiscuity produced by single substitutions in the enolase superfamily, Biochemistry 42, 8387-8393.
82. Yew, W. S., Fedorov, A. A., Fedorov, E. V., Rakus, J. F., Pierce, R. W., Almo, S. C., and Gerlt, J. A. (2006) Evolution of enzymatic activities in the enolase superfamily: L-Fuconate dehydratase from Xanthomonas campestris, Biochemistry 45, 14582-14597.
83. Yew, W. S., Fedorov, A. A., Fedorov, E. V., Wood, B. M., Almo, S. C., and Gerlt, J. A. (2006) Evolution of enzymatic activities in the enolase superfamily: D-Tartrate dehydratase from Bradyrhizobium japonicum, Biochemistry 45, 14598-14608.
84. Yew, W. S., Fedorov, A. A., Fedorov, E. V., Almo, S. C., and Gerlt, J. A. (2007) Evolution of enzymatic activities in the enolase superfamily: L-Talarate/galactarate dehydratase from Salmonella typhimurium LT2, Biochemistry 46, 9564-9577.
85. Gulick, A. M., Hubbard, B. K., Gerlt, J. A., and Rayment, I. (2000) Evolution of enzymatic activities in the enolase superfamily: Crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli, Biochemistry 39, 4590-4602.
86. Gulick, A. M., Palmer, D. R., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (1998) Evolution of enzymatic activities in the enolase superfamily: Crystal structure of D-glucarate dehydratase from Pseudomonas putida, Biochemistry 37, 14358-14368.
87. Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Vick, J. E., Babbitt, P. C., Almo, S. C., and Gerlt, J. A. (2007) Evolution of enzymatic activities in the enolase superfamily: D-Mannonate dehydratase from Novosphingobium aromaticivorans, Biochemistry 46, 12896-12908.
88. Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., and Kenyon, G. L. (1991) Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant, Biochemistry 30, 9274-9281.
89. Bone, R., Silen, J. L., and Agard, D. A. (1989) Structural plasticity broadens the specificity of an engineered protease, Nature 339, 191-195.
90. Aharoni, A., Gaidukov, L., Khersonsky, O., Gould, S. McQ, Roodveldt, C., and Tawfik, D. S. (2005) The 'evolvability' of promiscuous protein functions, Nat. Genet. 37, 73-76.
91. Yoshikuni, Y., Ferrin, T. E., and Keasling, J. D. (2006) Designed divergent evolution of enzyme function, Nature 440, 1078-1082.
92. Lassila, J. K., Keeffe, J. R., Kast, P., and Mayo, S. L. (2007) Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis, Biochemistry 46, 6883-6891.
93. Shao, W., Everitt, L., Manchester, M., Loeb, D. D., Hutchison, C. A., and Swanstrom, R. (1997) Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants, Proc. Natl. Acad. Sci. U.S.A. 94, 2243-2248.
94. Tozser, J., Yin, F. H., Cheng, Y. S., Bagossi, P., Weber, I. T., Harrison, R. W., and Oroszlan, S. (1997) Activity of tethered human immunodeficiency virus 1 protease containing mutations in the flap region of one subunit, Eur. J. Biochem. 244, 235-241.
95. Katoh, E., Louis, J. M., Yamazaki, T., Gronenborn, A. M., Torchia, D. A., and Ishima, R. (2003) A solution NMR study of the binding kinetics and the internal dynamics of an HIV-1 protease-substrate complex, Protein Sci. 12, 1376-1385.
96. Liu, F., Boross, P. I., Wang, Y. F., Tozser, J., Louis, J. M., Harrison, R. W., and Weber, I. T. (2005) Kinetic, stability, and structural changes in high-resolution crystal structures of HIV-1 protease with drug-resistant mutations L24I, I50V, and G73S, J. Mol. Biol. 354, 789-800.
97. Heaslet, H., Rosenfeld, R., Giffin, M., Lin, Y. C., Tam, K., Torbett, B. E., Elder, J. H., McRee, D. E., and Stout, C. D. (2007) Conformational flexibility in the flap domains of ligand-free HIV protease, Acta Crystallogr. D63, 866-875.
98. Miller, B. G. (2007) The mutability of enzyme active-site shape determinants, Protein Sci. 16, 1965-1968.
99. DeLano, W. L. (2007) MacPyMOL: A PyMOL-based Molecular Graphics Application for MacOS X, DeLano Scientific LLC, Palo Alto, CA.