Titration of ionizable groups in proteins using multiple neutron data sets from a single crystal: application to the small GTPase Ras.

ABSTRACT: Neutron protein crystallography (NPC) reveals the three-dimensional structures of proteins, including the positions of H atoms. The technique is particularly suited to elucidate ambiguous catalytic steps in complex biochemical reactions. While NPC uniquely complements biochemical assays and X-ray structural analyses by revealing the protonation states of ionizable groups at and around the active site of enzymes, the technique suffers from a major drawback: large single crystals must be grown to compensate for the relatively low flux of neutron beams. However, in addition to revealing the positions of hydrogens involved in enzyme catalysis, NPC has the advantage over X-ray crystallography that the crystals do not suffer radiation damage. The lack of radiation damage can be exploited to conduct in crystallo parametric studies. Here, the use of a single crystal of the small GTPase Ras to collect three neutron data sets at pD 8.4, 9.0 and 9.4 is reported, enabling an in crystallo titration study using NPC. In addition to revealing the behavior of titratable groups in the active site, the data sets will allow the analysis of allosteric water-mediated communication networks across the molecule, particularly regarding Cys118 and three tyrosine residues central to these networks, Tyr32, Tyr96 and Tyr137, with pKa values expected to be in the range sampled in our experiments.

SUBMITTER: Knihtila R 

PROVIDER: S-EPMC6360437 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature