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.Project 1: Mechanism of DNA helicase PcrAOver the past year, the Theoretical and Computational Biophysics Group has usedthe powerful Jonas cluster at PSC to investigate the mechanism of DNA unwindingin the DNA helicase PcrA from Bacillus stearothermophillus. The DNA unwindingaction of PcrA is caused by helicase translocation along single stranded DNA,which is driven by the hydrolysis of adenosine tri-phosphate (ATP) in a singlecatalytic site.We have used combined quantum mechanical/molecular mechanical (QM/MM)simulations to study the ATP hydrolysis reaction pathway and its coupling toprotein conformational changes in PcrA helicase. The simulation systemconsisted of about 20,000 atoms, 77 of which were treated quantum mechanicallyat the B3LYP/6-31G level of theory; the classical part was modeled using theAMBER94 force field. In order to simulate such a large QM/MM system, we reliedcrucially on the availability of the Jonas cluster at PSC with its large sharedmemory architecture, which allowed our code to run efficientlyon up to 32 processors.Analogous to our previous studies of ATP hydrolysis in F1-ATPase, a protonrelay mechanism was identified as the physiologically relevant proton transferpathway during nucleophilic attack. The 'arginine finger' residue R287 from aprotein domain neighboring the catalytic site was found to be crucial fortransition state stabilization, thereby, adding to the list of similaritiesbetween PcrA and F1-ATPase at the catalytic site level. Employing in silicomutation studies, it could be shown that the position of Q254 with respect tothe terminal phosphate group of ATP greatly influences the energy of the ATPhydrolysis product state, since a slight decrease in side chain length(mutation Q254N) changed the reaction energy profile from endothermic in thewild type to almost equi-energetic. This suggests a mechanism by which proteinconformational changes induced by DNA translocation are coupled to thecatalytic reaction in the binding site of PcrA.
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