Enzymes are frequently used in industrial settings and in applications such as pharmaceuticals and fuel production. However, many of these systems are not advantageous for proteins to flourish, with high temperatures and pH varying from that of most biological systems. Post-translational modifications are one method of improving characteristics of proteins such as thermal stability in these such environments. One example of these modifications, protein-polymer conjugates, exhibit greater enzymatic activity and stability in adverse conditions, however, the mechanism of interaction between the biomolecule and synthetic polymer is unknown. Speculation as to how this interaction occurs is undergoing debate as to whether the polymer interacts at the protein surface, stabilizing through noncovalent interactions, or extending out into solution, affecting the hydration sphere of the protein. Using a model protein, ubiquitin, and an N,N-dimethylacrylamide polymer with a spin label attached at various points, we aim to study this interaction through nuclear magnetic resonance (NMR) techniques highlighting the paramagnetic relaxation enhancement (PRE) effect. Furthermore, the polymer is able to conjugate with primary amines, found both in lysine residues and at the N-terminus. We have expressed and are purifying an engineered variant of ubiquitin with no lysine (K) residues present, dubbed K0 Ubiquitin, that theoretically only shows one possible conjugation site at the N-terminus. Using the K0 Ubiquitin variant, we will be able to further determine the method of interaction within the bioconjugate through more quantitative measurements and analysis.
Author: Jonathan Montgomery
Faculty Advisors: Richard C. Page and Dominik Konkolewicz, Dept. of Chemistry and Biochemistry
Graduate Student Advisors: Benjamin A. Shurina and Kevin Burridge, Dept. of Chemistry and Biochemistry
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