Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

Many signal transduction pathways are regulated by guanine nucleotide-binding (G?) proteins, which function as molecular switches fluctuating between active and inactive conformations. Proper function depends on three flexible switch regions that are involved in the relatively slow hydrolysis of GTP. Deep sequencing studies have found mutations in the GNAS and GNAI1 genes involved in tumorigenesis, among which include a mutation corresponding to a highly conserved arginine residue in the switch II region. A mutation in GNAI1 encoding an R208Q change in G?i1 has been linked to intestinal cancers. We investigated the molecular basis of oncogenesis of this mutant by studying the kinetics of nucleotide binding and single-turnover GTP hydrolysis. We demonstrated that, relative to the corresponding wild-type proteins, this mutation hindered nucleotide exchange; however, the rate of GTP hydrolysis was lower in R208Q G?i1. The R208Q G?i1 mutant was crystallized and its X-ray structure was compared to that of the wild-type protein and was used to conduct molecular dynamics simulations. Furthermore, we investigate the folding and structural integrity of the protein with three spectroscopic techniques. We show that both the active and inactive conformations of have similar melting temperatures, which are comparable to the inactive conformation of the wild-type protein and lower relative to the active conformation. These studies suggested that changes in the rates of hydrolysis can be attributed to alterations in the microenvironments of the nucleotide binding site which seemingly destabilize the switch II region but do not perturb the surface of the protein. The mutation presumably results in a decrease in the production of the secondary messenger cAMP via its interaction with the effector adenylyl cyclase that might promote oncogenesis in tumor cells.

Creative Commons License

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

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