Date of Award

Spring 2026

Abstract

The initial events in vision occur in the rod and cone photoreceptor cells of the retina, in which detection of light leads to an electrical response that occurs on the millisecond timescale. While the biochemical pathway of visual excitation is well understood, much less is known about the mechanism of the rapid recovery to the dark-adapted state. Physiological and biochemical studies have identified the protein complex that is the rate-limiting step for inactivation of the photoresponse: Regulator of G-protein Signaling 9-1 (RGS9-1), G-protein β-subunit5-Long (Gβ5L), and RGS9 Anchoring Protein (R9AP). The RGS9-1-Gβ5L-R9AP inactivation complex plays a crucial role in the visual transduction pathway by accelerating GTPase activity of the transducin α-subunit, thereby terminating activation of phosphodiesterase 6 (PDE6). This allows the pathway to recover to the dark-adapted state quickly. While an x-ray structure has been published depicting the interaction between RGS9-1 and Gβ5L, no structure of the inactivation complex with R9AP is available. The purpose of this research is to fill this gap in knowledge by analyzing the RGS9-1-Gβ5L-R9AP inactivation complex under near-physiological conditions. In the first stage of this work, the baculovirus expression system was used to express RGS9-1 and Gβ5L in insect cells; a second baculovirus expressing R9AP was also used for co-infection in order to express the heterotrimer. Recombinant R9AP was also expressed in bacteria. Proteins purified from these expression systems were reconstituted on liposomes to mimic the membrane-confined state of the inactivation complex in vivo. In the second phase of the work, the reconstituted, membrane-associated inactivation complex was exposed to chemical crosslinkers, the crosslinked proteins separated on polyacrylamide gels, and the isolated protein bands processed for mass spectrometry to identify cross-linked species. This data was used to perform integrative structural modeling of the inactivation complex, informed by the distance restraints imposed by the chemical crosslinks. Intra- and inter-molecular crosslinks involving RGS9-1 and Gβ5L permitted structural refinement of the x-ray structure of the heterodimer (PDB ID: 2PBI). Intramolecular crosslinks within R9AP provided experimental evidence for refining the AlphaFold-predicted structure of R9AP, while inter-molecular crosslinks between R9AP and RGS9-1 revealed major sites of interaction between the DHEX domain of RGS9-1 and the central α-helical bundle of R9AP. This work provides the first complete structure of the RGS9-1 inactivation complex of the visual transduction pathway and has clinical applications for understanding the pathogenicity of inherited mutations in the three genes of the inactivation complex that result in bradyopsia and other retinal diseases.

Document Type

Master's Thesis

First Advisor

Rick H. Cote

Second Advisor

Harish Vashisth

Third Advisor

Krisztina Varga

Department or Program

Biochemistry

Degree Name

Master of Science

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