This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Protein dynamics is known to play an important role in protein function. Certain level of internal mobility is required for proper function, and when proteins undergo so-called glass-transition, they lose the ability to bind substrates. Glass transition occurs universally at ~180K in many proteins, which together with the structural and computational data led to hypotheses that it is controlled by hydration shell, i.e. that protein dynamics is enslaved by solvent. Our preliminary study of protein dynamics in hyperthermophilic isopropylmalate dehydrogenase (IPMDH) at different temperatures indicates that glass-transition for this protein is shifted to ~250K. The proposed experiment includes careful examination of protein dynamics in IPMDH at atomic resolution at different temperatures in order to unravel the molecular mechanism of glass-transition in hyperthermophilic enzymes. Furthermore, we are interested in using a similar multi-temperature data collection strategy at atomic resolution to investigate the temperature dependence of protein disorder in an abortive ternary complex of E. coli dihydrofolate reductase (DHFR) bound to NADP+ and folate. Previous data collected from these crystals at 100 K extends to beyond 0.95 and indicates that several regions of the protein are disordered and that this disorder may be related to previously proposed catalytically relevant protein dynamics. However, establishing the functional relevance of this disorder requires diffraction data collected at a temperature at which the enzyme can catalyze hydride transfer.
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