Date of Award
Fall 2004
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Kincaid, James R.
Second Advisor
Steinmetz, Mark G.
Third Advisor
Sem, Daniel
Abstract
We have investigated ligand protein interactions for two major signaling proteins, glutamate receptors and soluble guanylyl cyclase, using various spectroscopic methods with the aim of understanding the ligand control of protein functions that play important roles in various physiological processes. Glutamate receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. They are essential in memory and learning, and play roles in a number of diseases and injury states. This work was aimed at gaining insight into the allosteric mode of function regulation in the glutamate receptors. We first used UV-Vis spectroscopy to clearly establish that the electronic environment of antagonists were similar in the isolated ligand binding domain of the receptor (S1S2) as in the native full receptors expressed in cells. Then the specific ligand protein interactions were characterized by using Fourier transform infrared (FTIR) spectroscopy. This allowed us to perform a time resolved study at the identified vibrations arising from the protein and ligand (glutamate) due to ligand binding. We focused on a mutant in which the kinetic steps were slower relative to the wild type receptor. These studies indicate that in the first step of ligand binding, the ligand glutamate docks at the α-carboxylate inducing large conformational changes in the protein (secondary structure), and the second step is established with small rearrangements resulting in the ligated state. Guanylate cyclase (sGC) converts GTP into second messenger cGMP upon activation by nitric oxide (NO). NO converts sGC into a high enzyme activity state, while carbon monoxide (CO) binding to sGC has little effect on the activity. The CO bound form exhibits high activity in the presence of allosteric effector, BAY-41-2272. Preliminary data indicated that the six coordinate heme in the CO-sGC, is converted to two new species, a five coordinate and a different six coordinate form. We have performed a systematic study of varying the concentration of BAY and temperature to determine the extent of the two forms by FTIR spectroscopy. These studies will be correlated to enzyme activity with the aim of obtaining insight into the role of these two species.