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
References: Not available.
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.