The long-term focus of this grant continues to be the structure and function of the visual pigment rhodopsin and its interactions with other members of the vertebrate phototransduction cascade. The studies are geared toward an understanding of the protein in terms of its mechanism of activation, its interaction with downstream proteins of the phototransduction cascade, and its function and dysfunction in health and disease. The new studies focus on two areas: the development and exploitation of a single-molecule approach to the study of interactions among signaling partners in the visual phototransduction cascade, and the characterization of novel mutants of rhodopsin in which the active site of the protein has been significantly altered from the wild-type. There are two Specific Aims: 1. to establish, characterize, and expand Total Internal Reflection Fluorescence Microscopy (TIRFM) as a tool in the single-molecule regime to study the dynamics of assembly and disassembly of key signaling complexes in the rod cell phototransduction cascade. One of the major goals of this aim is to better understand the role of constitutively active mutants in the diseases retinitis pigmentosa and congenital stationary night blindness. 2. To characterize novel rhodopsin mutants in which the highly conserved active-site Lys296 has been moved to different locations in the protein.
This Aim expands upon surprising preliminary results showing that the Lys can be moved while maintaining near wild-type spectral properties and ability to activate transducin in a light-dependent manner. The focus is to better understand the evolutionary relatedness of retinylidene proteins and to probe our understanding of the molecular mechanism of photoactivation of rhodopsin.

Public Health Relevance

The long-term focus of this grant is on the visual cascade of rod photoreceptor cells in the vertebrate retina, with particular emphasis on elucidation of the role of constitutively active mutants of the visual pigment rhodopsin in the diseases retinitis pigmentosa and congenital stationary night blindness.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY007965-25
Application #
8695851
Study Section
Special Emphasis Panel (BVS)
Program Officer
Neuhold, Lisa
Project Start
1988-12-01
Project End
2018-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
25
Fiscal Year
2014
Total Cost
$384,615
Indirect Cost
$134,615
Name
Brandeis University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
D'Antona, Aaron M; Xie, Guifu; Sligar, Stephen G et al. (2014) Assembly of an activated rhodopsin-transducin complex in nanoscale lipid bilayers. Biochemistry 53:127-34
Devine, Erin L; Oprian, Daniel D; Theobald, Douglas L (2013) Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins. Proc Natl Acad Sci U S A 110:13351-5
Deupi, Xavier; Edwards, Patricia; Singhal, Ankita et al. (2012) Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II. Proc Natl Acad Sci U S A 109:119-24
Standfuss, Jorg; Edwards, Patricia C; D'Antona, Aaron et al. (2011) The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471:656-60
Xie, Guifu; D'Antona, Aaron M; Edwards, Patricia C et al. (2011) Preparation of an activated rhodopsin/transducin complex using a constitutively active mutant of rhodopsin. Biochemistry 50:10399-407
Standfuss, Jorg; Xie, Guifu; Edwards, Patricia C et al. (2007) Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol 372:1179-88
Bayburt, Timothy H; Leitz, Andrew J; Xie, Guifu et al. (2007) Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins. J Biol Chem 282:14875-81
Tam, Beatrice M; Xie, Guifu; Oprian, Daniel D et al. (2006) Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis. J Neurosci 26:203-9
Xie, Guifu; Gross, Alecia K; Oprian, Daniel D (2003) An opsin mutant with increased thermal stability. Biochemistry 42:1995-2001
Jin, Shengnan; McKee, Timothy D; Oprian, Daniel D (2003) An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis. FEBS Lett 542:142-6

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