Investigation of protein-ligand interactions on a proteomic scale
Abstract
In this post-genomic and proteomic era, scientists are trying to study biological questions on a proteomic scale. The study of conformational energetics of protein folding on a proteomic scale is also a high potential method for addressing important questions and developing critical applications. Here I report a novel methodology to monitor changes in protein stability upon ligand binding on a proteomic scale by using a short pulse of proteolysis. Using this energetics-based identification approach, I have successfully identified ATP-binding proteins in E. coli proteome. Moreover, my proteomics screen revealed proteins that are not previously known to interact with ATP. I also confirmed the proteomics screen by determining the effect of ATP on the stabilities of some of the identified proteins. We believe this novel approach will be extremely valuable in identifying drug targets and off-targets as well as metabolite-binding proteins. While performing the proteomics screen, I have also observed an intriguing phenomenon that one of the identified proteins, glyceraldehyde-3-phosphate dehydrogenase, showed decreased apparent stability and increased unfolding rate in the presence of a physiological concentration of ATP. With further experiments, I successfully showed that ATP specifically stabilizes a dimeric unfolding intermediate of the enzyme. This result suggests that ATP may function as a chemical chaperone to facilitate the folding of this enzyme in E. coli. This finding is especially interesting because the interaction with endogenous metabolites has not been considered as a factor affecting protein folding in vivo. A new unfolding probe, absorbance at 230 nm (A230), is also reported here for the study of protein stability and kinetics of protein unfolding. Like absorbance at 280 nm, aromatic side chains, especially tryptophan, are known to contribute most to the change in A230. Our experiments clearly demonstrate that A230 is a suitable conformational probe of monitoring the unfolding of proteins in denaturants. The successful combination of A230 with 96-well microtiter plates further shows the feasibility on studying protein stability and kinetics of protein unfolding on a high-throughput manner.
Degree
Ph.D.
Advisors
Park, Purdue University.
Subject Area
Biochemistry|Biophysics
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