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STRUCTURAL AND BIOCHEMICAL STUDIES OF RPE65, THE RETINOID ISOMERASE OF THE VISUAL CYCLE

Kiser, Philip David
http://rave.ohiolink.edu/etdc/view?acc_num=case1269869379

Abstract Details

2010, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium. Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis. Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Additionally, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To obtain structural insights into these questions the crystal structure of RPE65 from bovine retinal pigment epithelium was determined. The structure reveals the architecture of the enzymatic active site and a hydrophobic patch that likely mediates membrane binding. Extraction and phase separation experiments indicate a direct interaction between hydrophobic side chains of RPE65 and the acyl core of the lipid bilayer consistent with crystal structure. The structure also provides insights into the mechanisms by which Leber congenital amaurosis-associated RPE65 amino acid substitutions lead to loss of function. To facilitate characterization of RPE65 a monoclonal antibody was generated and its epitope was mapped within the RPE65 sequence. The antibody specifically stains retinal pigment epithelium cells in retinal sections and is useful for immunoaffinity purification of RPE65 and RPE65-containing complexes. The studies described here provide key insights into the biology and biochemistry of this important visual cycle enzyme.
Palczewski Krzysztof, PhD (Advisor)
Anthony Berdis, PhD (Committee Chair)
Michael Maguire, PhD (Committee Member)
Focco van den Akker, PhD (Committee Member)
167 p.
Biochemistry
Retinoids; monotopic membrane protein; isomerase; metalloprotein

Recommended Citations

Citations

  • Kiser, P. D. (2010). STRUCTURAL AND BIOCHEMICAL STUDIES OF RPE65, THE RETINOID ISOMERASE OF THE VISUAL CYCLE [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1269869379

    APA Style (7th edition)

  • Kiser, Philip. STRUCTURAL AND BIOCHEMICAL STUDIES OF RPE65, THE RETINOID ISOMERASE OF THE VISUAL CYCLE. 2010. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1269869379.

    MLA Style (8th edition)

  • Kiser, Philip. "STRUCTURAL AND BIOCHEMICAL STUDIES OF RPE65, THE RETINOID ISOMERASE OF THE VISUAL CYCLE." Doctoral dissertation, Case Western Reserve University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1269869379

    Chicago Manual of Style (17th edition)

case1269869379
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© 2010, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.