Poster Session I
Project Type
Poster
Project Funding and Affiliations
The Northern Rockies Center for Hydrodynamicsis supported by the NIH CoBRE and NIH grant S10-OD036366, The Integrated Structural Biology Core of the Center for Biomolecular and Structural Dynamics is supported by NIGMS grant P30GM140963
Faculty Mentor’s Full Name
Borries Demeler
Faculty Mentor’s Department
Chemistry/BioChemistry
Additional Mentor
Michelle Nemetchek, Cindee Yates, Live McClelland
Abstract / Artist's Statement
G-protein coupled receptors (GPCRs) are a target of 35% of FDA-approved drugs and play a major role in cellular communication. GPCRs activate various Gα proteins, such as Gαi, which activate downstream effector proteins and result in intracellular signaling. Ric-8A is a molecular chaperone protein required for Gαi biogenesis and has cytosolic guanine nucleotide exchange factor activity. Understanding proteins which regulate GPCR pathways, like Ric-8A, may reveal novel therapeutic targets in G-protein signaling.
As an undergraduate volunteer in the Analytical Ultracentrifugation (AUC) core, my goal is to use AUC to characterize the binding behavior of Gαi and Ric-8A, independently verify association constants found using isothermal titration calorimetry, and compare this behavior to mutant Ric-8A proteins. This will provide important information on the binding surface, mechanism, and character of the Gαi- Ric-8A interaction. We hypothesize that Gαi and Ric-8A bind with micromolar range affinity and mutations on the binding surface of Ric-8A will disrupt this interaction.
In AUC, individual molecules in mixtures are hydrodynamically separated under extreme centrifugal force and identified by their rate of sedimentation. Preliminary experiments included collecting UV-Vis spectra of Gαi, GDP, and Ric-8A at various concentrations to create a reference dataset. Our initial AUC experimental results indicate an increase in binding at increasing ratios of Gαi:Ric-8A. At a 4:1 ratio, excess Gαi is observed. This preliminary work verifies our method and the purity of our proteins. Our future AUC work will evaluate Gαi and wildtype/mutant Ric-8A at physiologically relevant concentrations and define essential amino acids on their interaction surface.
Category
Physical Sciences
Using Analytical Ultracentrifugation to Characterize Ric8:Gαi Protein Binding
UC South Ballroom
G-protein coupled receptors (GPCRs) are a target of 35% of FDA-approved drugs and play a major role in cellular communication. GPCRs activate various Gα proteins, such as Gαi, which activate downstream effector proteins and result in intracellular signaling. Ric-8A is a molecular chaperone protein required for Gαi biogenesis and has cytosolic guanine nucleotide exchange factor activity. Understanding proteins which regulate GPCR pathways, like Ric-8A, may reveal novel therapeutic targets in G-protein signaling.
As an undergraduate volunteer in the Analytical Ultracentrifugation (AUC) core, my goal is to use AUC to characterize the binding behavior of Gαi and Ric-8A, independently verify association constants found using isothermal titration calorimetry, and compare this behavior to mutant Ric-8A proteins. This will provide important information on the binding surface, mechanism, and character of the Gαi- Ric-8A interaction. We hypothesize that Gαi and Ric-8A bind with micromolar range affinity and mutations on the binding surface of Ric-8A will disrupt this interaction.
In AUC, individual molecules in mixtures are hydrodynamically separated under extreme centrifugal force and identified by their rate of sedimentation. Preliminary experiments included collecting UV-Vis spectra of Gαi, GDP, and Ric-8A at various concentrations to create a reference dataset. Our initial AUC experimental results indicate an increase in binding at increasing ratios of Gαi:Ric-8A. At a 4:1 ratio, excess Gαi is observed. This preliminary work verifies our method and the purity of our proteins. Our future AUC work will evaluate Gαi and wildtype/mutant Ric-8A at physiologically relevant concentrations and define essential amino acids on their interaction surface.