Picosecond-ResolvedFluorescence Studies of Substrateand Cofactor-Binding Domain Mutants in a Thermophilic Alcohol DehydrogenaseUncover an Extended Network of Communication

摘要

Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 Å apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-exposed site (Trp167), time-dependent Stokes shifts and collisional quenching data allow an analysis of intra-protein dynamical communication. A double mutant, Y25A:V260A, was also inserted into each single-Trp construct and analyzed accordingly. None of the mutations affect fluorescence lifetimes, Stokes shift relaxation rates, and quenching data for the surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent probes of the active-site tryptophan 87 reveal distinctive forms of dynamical communication. Stokes shifts show that the distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process not previously reported by such measurements,and the double mutant (Y25A:V260A) eliminates the temperature-dependenttransition sensed by the active-site tryptophan in the presence ofV260A. Collisional quenching data at Trp87 further show a structuralchange in the active-site environment/solvation for V260A. In theaggregate, the temperature dependencies of the fluorescence data aredistinct from the breaks in behavior previously reported for catalysisand hydrogen/deuterium exchange, attributed to time scales for theinterconversion of protein conformational substates that are slowerand more global than the local motions monitored within. An extendednetwork of dynamical communication between the protein dimer surfaceand substrate- and cofactor-binding domains emerges from the flourescentdata.