Studies of macromolecular crowding

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In collaboration with the group of E. Haas we have investigated the effect of a small "inert" cosolute, trimethylamine oxide, on the conformational isomerization of an enzyme, adenylate kinase, that is known to undergo significant conformational changes during its catalytic cycle. Binding of substrate nucleotides (AMP, ADP, ATP, or analogs thereof) is accompanied by compaction resulting from the closure of clefts containing ligand binding sites. Conformational changes are being characterized via time-dependent fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores placed at selected locations along the peptide chain through site-specific mutagenesis. Analysis of the results obtained to date indicate that sufficient concentrations of TMAO stabilize compact or "closed" conformations relative to less compact or "open" conformations, leading to a substantial increase in the affinity of the enzyme for ligands. A report of this work was recently published.