Keywords: Atomic Resolution, Crystal Structure, Pk(a), Rnase A, Titration, Histidine, Protein, Ribonuclease A, Article, Enzyme Active Site, Enzyme Structure, Priority Journal, Protein Structure, Proton Transport, Reaction Analysis, Stereochemistry, Titrimetry, X Ray Diffraction, Binding Sites, Crystallization, Crystallography, X-Ray, Electrons, Electrostatics, Hydrogen Bonding, Hydrogen-Ion Concentration, Models, Molecular, Protein Conformation, Pancreatic, Pk (a),
Affiliations: Ctro. Stud. Biocristallografia D., Univ. di Napoli Federico II, via Mezzocannone 4, I-80134, Napoli, Italy
Europ. Molec. Biol. Lab. (EMBL), c/o DESY Notkestrasse 85, D-22603, Hamburg, Germany
Department of Chemistry, University of York, Heslington, York, YO1 5DD, United Kingdom
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Walsh, M.A., Schneider, T.R., Sieker, L.C., Dauter, Z., Lamzin, V.S., Wilson, K.S., Refinement of triclinic hen egg white lysozyme at atomic resolution (1998) Acta Crystallog. Sect. D, 54, pp. 522-546
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Wlodawer, A., Sjölin, L., Structure of ribonuclease A: Results of joint neutron and X-ray refinement at 2.0 Å resolution (1983) Biochemistry, 22, pp. 2720-2728
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Baker, W. R., Kintanar, A., Characterisation of the pH titration shifts of ribonuclease A by one- And two-dimensional nuclear magnetic resonance spectroscopy (1996) Arch. Biochem. Biophys., 327, pp. 189-19
Chayen, N. E., The role of oil in macromolecular crystallisation (1997) Structure, 5, pp. 1269-1274
Cuchillo, C. M., Vilanova, M., Nogu s, M. V., Pancreatic ribonucleases (1997) Ribonucleases: Structures and Functions, pp. 271-304. , G. D'Alessio, & J. F. Riordan. London: Academic Press
De Mel, V. S. J., Doscher, M. S., Martin, P. D., Edwards, B. F. P., The occupancy of two distinct conformations by active-site histidine-119 in crystals of ribonuclease is modulated by p H (1994) FEBS Letters, 349, pp. 155-160
Dunitz, J. D., Shomaker, V., Trueblood, K. N., Interpretation of atomic displacement parameters from diffraction studies of crystals (1988) J. Phys. Chem., 92, pp. 856-867
Ebina, S., W trich, K., Amide proton titration shifts in bull seminal inhibitor IIA by two-dimensional correlated1H nuclear magnetic resonance (COSY) (1984) J. Mol. Biol., 179, pp. 283-288
Engh, R. A., Huber, R., Accurate bond and angle parameters for X-ray protein structure refinement (1991) Acta Crystallog. Sect. a, 47, pp. 392-400
Garman, E. F., Schneider, T. R., Macromolecular cryocrystallography (1997) J. Appl. Crystallog., 30, pp. 211-237
Gilliland, G. L., Crystallographic studies of ribonuclease complexes (1997) Ribonucleases: Structures and Functions, pp. 305-341. , G. D'Alessio, & J. F. Riordan. London: Academic Press
Harrington, W. F., Schellman, J. A., Evidence for the instability of hydrogen-bonded peptide structures in water, based on studies of ribonuclease and oxidized ribonuclease (1956) C. R. Trav. Lab. Carlsberg, 30, pp. 21-43
Jeffery, G. A., Saenger, W., (1981) Hydrogen Bonding in Biological Structures, , Berlin: Springer Verlag
Kim, E. E., Varadarajan, R., Wyckoff, H. W., Richards, F. M., Refinement of the crystal structure of ribonuclease S. Comparison with and between the various ribonuclease A structures (1992) Biochemistry, 31, pp. 12304-12314
Li, J. R., Waltz, F. G., A steady state kinetic study of the ribonuclease A catalyzed hydrolysis of Uridine-2 -3 (cyclic) -5 -diphosphate (1974) Arch. Biochem. Biophys., 161, pp. 227-233
Martin, P. D., Dosher, M. S., Edwards, B. F. P., The refined crystal structure of a fully active semisinthetic ribonuclease at 1. 8 resolution (1987) J. Biol. Chem., 262, pp. 15930-15938
Sheldrick, G. M., Phase annealing in SHELX-90: Direct methods for larger structures (1990) Acta Crystallog. Sect. a, 46, pp. 467-473
Sheldrick, G. M., Schneider, T. R., SHELXL: High-resolution refinement (1997) Methods Enzymol., 277, pp. 319-343
Tilton, R. F., Dewan, J. C., Petsko, G. A., Effects of temperature on protein structure and dynamics: X-ray crystallographic studies of the protein Ribonuclease-A at nine different temperatures from 98 to 320 K (1992) Biochemistry, 31, pp. 2469-2481
Walsh, M. A., Schneider, T. R., Sieker, L. C., Dauter, Z., Lamzin, V. S., Wilson, K. S., Refinement of triclinic hen egg white lysozyme at atomic resolution (1998) Acta Crystallog. Sect. D, 54, pp. 522-546
Wilson, A. J. C., Determination of absolute from relative X-ray intensities (1942) Nature, 150, pp. 151-152
Wilson, K. S., Butterworth, S., Dauter, Z., Lamzin, V. S., Walsh, M., Wodak, S., Pontius, J., MacArthur, M. W., Who checks the checkers? (1998) J. Mol. Biol., 276, pp. 417-436
Wlodawer, A., Sj lin, L., Structure of ribonuclease A: Results of joint neutron and X-ray refinement at 2. 0 resolution (1983) Biochemistry, 22, pp. 2720-2728
Protein titration in the crystal state
Proteins are complex structures whose overall stability critically depends on a delicate balance of numerous interactions of similar strength, which are markedly influenced by their environment. Here, we present an analysis of the effect of pH on a protein structure in the crystalline state using RNase A as a model system. By altering only one physico-chemical parameter in a controlled manner, we are able to quantify the structural changes induced in the protein. Atomic resolution X-ray diffraction data were collected for crystals at six pH* values ranging from 5.2 to 8.8, and the six independently refined structures reveal subtle, albeit well-defined variations directly related to the pH titration of the protein. The deprotonation of the catalytic His12 residue is clearly evident in the electron density maps, confirming the reaction mechanism proposed by earlier enzymatic and structural studies. The concerted structural changes observed in the regions remote from the active-site point to an adaptation of the protein structure to the changes in the physico-chemical environment. Analysis of the stereochemistry of the six structures provided accurate estimates of pK(a), values of most of the histidine residues. This study gives further evidence for the advantage of atomic resolution X-ray crystallographic analyses for revealing small but significant structural changes which provide clues to the function of a biological macromolecule.