Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

Guanine nucleotide-binding (G?) proteins have been intensely studied over the years due to their role as signal transduction proteins. Proper function depends on three flexible switch regions that are involved in the relatively slow hydrolysis of GTP, allowing for the proteins to act as a molecular on/off switch. The structure of a protein is essential to its biological function. Occurrences of mutations in the genes GNAS and GNAI1 to highly conserved arginine residue in the switch II region have been detected in several types of cancers.

Utilizing the combinations of temperature denaturation, kinetics assays, and molecular dynamics, we plan to study the consequences of these mutations. In this work, we probed the accessibility of buried Trp, solvent-exposed Tyr residues, as well as determine the secondary structure content of wild-type and Trp/Arg mutants under denaturating conditions. Furthermore, we investigated the ?-cation interactions between Trp and Arg residues in G? proteins as unfolding occurs and ascertain how the oncogenic Gs? R231H mutation results in a decrease in signal transduction efficiency compared to wild-type. We demonstrated that, relative to the corresponding wild-type proteins, destabilization of the non-covalent interaction is limited to the active conformation. Furthermore, although the mutation occurs at the same position and both Gi?1 and Gs? are structurally similar, the mutation results in drastically different structural changes. The kcat value for the R208Q Gi?1 mutant was lower than for the WT Gi?1 subunit, whereas, for the R231H Gs? mutant, the rate of GTP hydrolysis was higher than for the WT Gs? protein, indicating different mechanism of action. Although the effects on hydrolysis are different, both mutations presumably result in a decrease in the production of cAMP that might promote oncogenesis in tumor cells.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Available for download on Wednesday, October 11, 2023

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