Protein dynamics: Studies of adenylate kinase mutants from Escherichia coli and characterization of adenylate kinase isoforms from murine cells. Applications of fluorescence spectroscopy and microscopy

The first section of the thesis is focused on the fluorescence spectroscopic studies on the folding and dynamics of adenylate kinase (AKe) mutants from E. coli. We have constructed a series of single trp mutants with the mutations distributed in various regions of the enzyme. Careful characterization of each trp mutant suggested the trp replacement had little effect on its overall structure and enzymatic function. The trp residue of each AKe mutant acted as a fluorescence reporter which allowed us to follow the local environmental changes during its folding process. A model of protein folding was suggested based on the correlation of the folding kinetics of AKe and the recovery of the enzymatic activity.; The second section of the thesis is focused on the characterization of two adenylate kinase isoforms (AK1 & AK1beta) from murine cells using a series of fluorescence microscopic techniques (two-photon excitation fluorescence imaging, fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging (FLIM)). In the in vivo studies, the genes of AK1 and AK1beta were fused with the gene of enhanced green fluorescence protein (EGFP) and then expressed in HeLa cells. Fluorescence images of the transfected cell showed that the AK1-EGFP was homogeneously distributed throughout the cell, while AK1beta-EGFP was mainly localized on the plasma membrane. The diffusion rates of AK1-EGFP and AK1beta-EGFP obtained from FCS measurements suggested that AK1 diffused freely in the cytosol of the cell, while AK1beta was bound to the plasma membrane. It has been suggested that AK1beta interacts with the plasma membrane through a myristoyl chain. To test the hypothesis, in the in vitro studies, AK1beta were over-expressed in E. coli, purified and tested for its interaction with a model membrane system (giant unilamellar vesicles (GUVs)). AK1beta showed no binding affinity to GUVs made from POPC in the fluorescence imaging study. However, when AK1beta was co-expressed with N-myristoyl-transferase (NMT), an enzyme that can myristoylate proteins with the proper signal in E. coli, the purified protein named as AK1betamyr showed binding affinity to the GUVs. This result suggested that myristoylation of AK1beta played an important role in its binding to the GUVs.