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A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8
Author(s) -
Merkley Eric D.,
Daggett Valerie,
Parson William W.
Publication year - 2012
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.23219
Subject(s) - thermus thermophilus , flavin group , conformational change , chemistry , flavin adenine dinucleotide , circular dichroism , cofactor , active site , fluorescence , flavoprotein , photochemistry , tryptophan , molecular dynamics , crystallography , stereochemistry , enzyme , biochemistry , computational chemistry , amino acid , physics , escherichia coli , quantum mechanics , gene
Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.