PROTEIN TITRATION IN THE CRYSTAL STATE

Rita Berisio^1,2 , Victor S. Lamzin², Filomena Sica¹, Keith S. Wilson³,Adriana Zagari¹, Lelio Mazzarella¹

1 Centro di Studio di Biocristallografia and Dipartimento di Chimica, Università di Napoli "Federico II", via Mezzocannone, 4; I-80134 Napoli, Italy.
2 European Molecular Biology Laboratory (EMBL) c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany.
3 Department of Chemistry, University of York, Heslington, York YO1 5DD, UK.

Biological sciences have gained many insights from the use of macromolecular X-ray crystallography. Recently, structures to an extremely high resolution (about 1 Å) have started to emerge, thus providing an exceptionally high level of details. These models are, in fact, able to unravel fine structural features which may be extremely relevant to the protein function. Although the accuracy of atomic resolution protein structures is comparable to the one typically achieved for small molecules, new issues can be addressed for proteins. In fact, the presence of liquid solvent in protein crystals allows to manipulate the physicochemical properties of the protein, by changing the properties of the medium. Therefore, processes where the proton transfer plays a crucial role, or where the ligand-binding affinity is regulated by pH, temperature or ionic strength, can be studied.

In this framework, we used X-ray synchrotron radiation to elucidate how the pH affects a protein structure, with the purpose of highlighting the role of the proton transfer in a biological process. Bovine pancreatic ribonuclease was chosen for this study, as it shows a pH dependent reaction mechanism and its crystals remain stable over a pH range wide enough to allow the change of the protonation state of the residues most deeply involved in the catalytic mechanism. The results of this study will be presented.