Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication.

ABSTRACT: 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.

SUBMITTER: Meadows CW 

PROVIDER: S-EPMC4210157 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature