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 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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Ruan, Qiaoqiao

Contributors dc:contributor

• William W. Mantulin
• Glaser, Michael

• Biophysics, General

Language dc:language

eng

Identifier

(MiAaPQ)AAI3070044

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/84784