Spectroscopic studies of protein folding and protein dynamics using single-tryptophan mutants of E. coli adenylate kinase.

摘要

Comprehensive studies of protein dynamics and equilibrium protein denaturation are undertaken utilizing a combined approach of site-directed mutagenesis and optical spectroscopic techniques. The enzyme adenylate kinase from the organism E. coli serves as the system to be studied, having been chosen as an ideal candidate for site-directed, single-tryptophan-substitution mutations. A set of six such single-tryptophan mutants of adenylate kinase are studied in depth, using steady-state and time-resolved fluorescence techniques, as well as circular dichroism spectroscopy. Particular mutants are chosen for investigation only if their global secondary structure and enzymatic activities do not differ significantly from those of the wild-type protein.;Following the initial spectroscopic characterization of the six single-tryptophan mutants at room temperature and neutral pH (both in the absence and presence of ligand), the mutants are studied exhaustively under conditions of high concentration of chemical denaturant, acidic pH, high temperature, and finally, high hydrostatic pressure--all at thermodynamic equilibrium. The structure and dynamics of the individual mutants under these various conditions are investigated using fluorescence spectroscopic techniques. Also, and of most importance, the interpretation of the results assumes that the tryptophans act as local reporter groups of the various tertiary micoenvironments of the wild-type enzyme. This provides a justification for modeling the non-native equilibrium states of the wild-type adenylate kinase.;Finally, native states of the mutants are studied at high concentrations of stabilizing cosolvent, both at room temperature and liquid-nitrogen temperatures. These studies provide insight into the native-state dynamics of the protein which are impossible to observe with the given techniques at room temperature in buffer. Additionally, the stabilizing cosolvents are observed to modify the tertiary structure of the enzyme even while leaving its secondary structure and ligand-binding capacity unaffected.;An "atlas" of the various equilibrium states of E. coli adenylate kinase is presented in the final summary chapter.