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 a re markedly influenced by their environment. Here, we present an analysis o f the effect of pH on a protein structure in the crystalline state using RN ase A as a model system. By altering only one physico-chemical parameter in a controlled manner, we are able to quantify the structural changes induce d in the protein. Atomic resolution X-ray diffraction data were collected f or crystals at six pH* values ranging from 5.2 to 8.8, and the six independ ently refined structures reveal subtle, albeit well-defined variations dire ctly related to the pH titration of the protein. The deprotonation of the c atalytic His12 residue is clearly evident in the electron density maps, con firming the reaction mechanism proposed by earlier enzymatic and structural studies. The concerted structural changes observed in the regions remote f rom the active-site point to an adaptation of the protein structure to the changes in the physico-chemical environment. Analysis of the stereochemistr y of the six structures provided accurate estimates of pK(a) values of most of the histidine residues. This study gives further evidence for the advan tage of atomic resolution X-ray crystallographic analyses for revealing sma ll but significant structural changes which provide clues to the function o f a biological macromolecule. (C) 1999 Academic Press.