Scotopic vision is initiated upon capture of a photon of light by rhodopsin molecules present in rod photoreceptor cells. The activation of the light receptor rhodopsin sets into motion a series of biochemical reactions called phototransduction, which leads to the hyperpolarization of the cell. The long-term goal of this research program is to understand the molecular mechanisms underlying the biochemical events in phototransduction under normal and diseased states. The starting point will be structure-function studies of rhodopsin. The importance of this molecule extends beyond its central role in phototransduction. The rhodopsin gene is a hot spot for mutations causing inherited vision disorders and these mutations are the leading cause of autosomal dominant retinitis pigmentosa, a heterogeneous group of inherited retinal degenerative diseases. Despite the wealth of knowledge available for rhodopsin, an accurate mechanism of its action is still unavailable and the mechanism underlying mutations in the light receptor causing vision disorders is unclear. Our immediate goal is to explore emerging ideas about the system that expand on classical dogma;namely, the notion of multiple active states of rhodopsin and the organization of rhodopsin into clusters of dimers.
The aims of the proposal are thematically linked around understanding the fundamental molecular principles governing the activity of rhodopsin in normal and diseased conditions in people. In the first aim, we will test the implicit assumption made in most studies that the structure and function of human rhodopsin is similar to that of the receptor from better-studied mammalian species (bovine and mouse) used to understand human disease pathology. In the second aim, we will test the hypothesis that there are multiple active states of the receptor and that at least one of these states leads to constitutive activity in a rhodopsn mutant causing congenital stationary night blindness. In the third aim, we will test a putative rhodopsin dimer model and determine whether receptor oligomerization contributes to the phenotype of a rhodopsin mutant causing autosomal dominant retinitis pigmentosa. Significant technological advances are required to overcome the intrinsic difficulties in studying membrane proteins to observe native structural and molecular details that are important to understand the system. Our proposal utilizes several high-resolution biophysical methods including atomic force microscopy, single-molecule force spectroscopy and Forster resonance energy transfer. The combination of these methods with more traditional biochemical, biophysical, and genetic approaches will overcome the limitations of traditional assays alone and allow us to directly test emerging paradigms about rhodopsin structure and function. The successful testing of these new concepts will lead to a more accurate molecular framework to understand the function of the system under normal conditions and dysfunctions in inherited human disease.
We are in an era of research where technology is giving us access to the smallest details of biological systems. Our proposal takes advantage of these technological advances in order to understand the initial events in vision under normal and diseased states. Our studies will reveal fundamental insights about the visual system and will reveal novel strategies to combat retinal disease.
|Senapati, Subhadip; Gragg, Megan; Samuels, Ivy S et al. (2018) Effect of dietary docosahexaenoic acid on rhodopsin content and packing in photoreceptor cell membranes. Biochim Biophys Acta Biomembr 1860:1403-1413|
|Gragg, Megan; Park, Paul S-H (2018) Misfolded rhodopsin mutants display variable aggregation properties. Biochim Biophys Acta Mol Basis Dis 1864:2938-2948|
|Rakshit, Tatini; Senapati, Subhadip; Parmar, Vipul M et al. (2017) Adaptations in rod outer segment disc membranes in response to environmental lighting conditions. Biochim Biophys Acta Mol Cell Res 1864:1691-1702|
|Mishra, Ashish K; Gragg, Megan; Stoneman, Michael R et al. (2016) Quaternary structures of opsin in live cells revealed by FRET spectrometry. Biochem J 473:3819-3836|
|Gragg, Megan; Kim, Tae Gyun; Howell, Scott et al. (2016) Wild-type opsin does not aggregate with a misfolded opsin mutant. Biochim Biophys Acta 1858:1850-9|
|Han, Xiangzi; Tang, Jinshan; Wang, Jingna et al. (2016) Conformational Change of Human Checkpoint Kinase 1 (Chk1) Induced by DNA Damage. J Biol Chem 291:12951-9|
|Miller, Lisa M; Gragg, Megan; Kim, Tae Gyun et al. (2015) Misfolded opsin mutants display elevated ?-sheet structure. FEBS Lett 589:3119-25|
|Park, Paul S-H; Müller, Daniel J (2015) Dynamic single-molecule force spectroscopy of rhodopsin in native membranes. Methods Mol Biol 1271:173-85|
|Rakshit, Tatini; Senapati, Subhadip; Sinha, Satyabrata et al. (2015) Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis. PLoS One 10:e0141114|
|Whited, Allison M; Park, Paul S-H (2015) Nanodomain organization of rhodopsin in native human and murine rod outer segment disc membranes. Biochim Biophys Acta 1848:26-34|
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