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Date of Award


Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science


Biochemistry & Molecular Biol.

First Advisor

Dr. Ellis Bell


Collisional quenching approaches using neutral or charged quencher molecules have long been used to indicate conformational changes in proteins. Here we introduce a novel approach to collisional quenching using multi-wavelength analysis of spectral data collected at two different excitation wavelengths with a variety of small molecule and protein test fluorophores. The results show that not only can detailed information about the local environment of tryptophans in proteins be obtained (even proteins with multiple tryptophans), but information is derived resulting from tyrosine-tryptophan resonance energy transfer in proteins. Using Acrylamide, Iodide and Cesium collisional quenchers with both tyrosine and tryptophan derivatives and with model proteins with one or more tryptophans, we demonstrate the utility of the approach for defining the local charge environment and changes induced by ligand binding, Furthermore, during the course of these studies we revealed that pi-cation effects disrupt resonance energy transfer between tryptophan (acting as either donor or acceptor) and other moieties in the protein. With Glutamate Dehydrogenase, we have used this technique to investigate substrate, cofactor and regulatory ligand interactions and found that ligands which support or promote activity lead to increased flexibility of the protein while ligands that inhibit the reaction have the opposite effect.