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Date of Award

Spring 2012

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science


Biochemistry & Molecular Biol.

First Advisor

Dr. Ellis Bell


3-Phosphoglycerate Dehydrogenase is a homotetramer with three types of subunit interfaces; between adjacent regulatory domains, cofactor domains, and across the central cavity of the tetramer. A single tryptophan per subunit lies at the cofactor interface and crosses over between adjacent subunits. Collisional quenching approaches using steady-state and lifetime measurements with both charged and neutral quenchers have been used to investigate the effects of ligands on solvent exposure and environment of this interface in native enzyme and a truncation mutant lacking the serine binding domain. NADH and α-ketoglutarate decrease overall accessibility of the tryptophan possibly through the rotation of an interface spanning loop while serine primarily affects the charge environment around the tryptophan. Rotation of this loop may orient residues at the interface to form cross interface interactions necessary for communication. A K141Q/L mutations shows a decrease in acrylamide quenching as well as a reversal in iodide and cesium quenching. Overall ligand binding to K141Q/L has less effect on quenching than in the native. This suggests ligand binding has less of a conformational effect than in the native. Thermal melts indicate a decrease in stability in these mutants while the minimal effects of ligands suggests changes in flexibility. Lifetime measurements indicate upward curvature seen in some Stern-Volmer plots results from static quenching which may be caused by asymmetry in the interface.