The proposed investigation seeks to apply phase fluorometry to an analysis of the interdependency of the tertiary and quaternary protein structure of the dimeric isozymes of creatine kinase. The fundamental question addressed is the nature of the interactions between subunit folding and subunit organization in the overall conformation of non-allosteric, multimeric proteins. Recent advances in fluorometric analysis provide sensitive and searching quantitative criteria by which to compare specific structural components including site-site distances, protein rotational rates, asymmetric subunit organization and motional freedom within the active site domain. Kinetics of subunit folding and subunit association will be analyzed by continuous measurement of resonance energy transfer efficiency. Lifetime measurements and time resolved anisotropy decay using phase/modulation fluorometry, constitute essential analytical procedures. Taken together, the procedures constitute an experimental approach to evaluating the contribution of tertiary and quaternary structure in the formation and maintenance of three-dimension conformation and response to interaction with substrate and substrate analog by creatine kinase. While used as a model for subunit interactions in non-allosteric oligomeric enzymes, creatine kinase exhibits several interesting features and is an important enzyme to study because of its central role in neuromuscular energy metabolism. The inquiry is representative the long term goal of better understanding the interdependency of subunit and oligomer structure in non-allosteric catalysis, regulation, compartmentation and stabilization essential for biological function.
| Grossman, S H (1990) Resonance energy transfer between the active sites of creatine kinase from rabbit brain. Biochim Biophys Acta 1040:276-80 |
| Grossman, S H; Akinade, F A; Garcia-Rubio, L (1990) A physicochemical comparison of the isozymes of creatine kinase from rabbit brain and muscle. Biochim Biophys Acta 1040:311-6 |
| Grossman, S H (1989) Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence. Biochemistry 28:4894-902 |
