Basic research on protein structure, function, and regulation has produced the following results: (1) Heat-induced unfolding of dodecameric Mn.glutamine synthetase (Mn.GS) from E. coli at Ph 7 has been studied by spectral techniques and by differential scanning calorimetry (DSC). In the absence of denaturant, cooperative interactions link partial, reversible unfolding reactions of all subunits within the GS dodecamer; mid-point T = 51.6 deg C. Urea destabilizes the enzyme and allows DSC measurements of dissociation and unfolding reactions, which occur at 50-95 deg C in 2 M urea. (2) Thermal denaturation of the tryptophan synthase (TS) complex (+/-) bound pyridoxal phosphate) from S. typhimurium also is being studied. DSC and spectral data are consistent with a model of 6 sequential two- state transitions for the TS alphabetabeta alpha complex: 3 transitions at <65 deg C involve exposure of Trp177 in beta chains and unfolding of 2 alpha chains and 3 at >70 deg C occur as beta chains in TS unfold. The cofactor increases both the linkage between unfolding domains and the stability of beta subunits. (3) Phospho- and dephospho- myosin-II from Acanthamoeba have identical DSC profiles with a single sharp endotherm at about 42 deg C. Work is in progress to identify thermodynamic domains. (4) Isothermal titration calorimetry of the catalytic (c) trimer of E. coli aspartate transcarbamoylase with a Zn-domain C-terminal regulatory (r) fragment indicate that a strong energetic coupling exists between substrate binding and r:c contact interactions. (5) Thermodynamic characterization of the influenza virus hemagglutinin reveals proton-induced conformational changes which may relate to physiological function. (6) Fluorescence studies on the interaction of transcriptional factor 111A with DNA fragments containing the inner control region of the 5S RNA gene are nearly complete.
