May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
CRALBP Topological Analyses by Mass Spectrometry
Author Affiliations & Notes
  • J.W. Crabb
    Cole Eye Institute, The Cleveland Clinic Foundation i31, Cleveland, OH, United States
  • A. Hasan
    Cole Eye Institute, The Cleveland Clinic Foundation i31, Cleveland, OH, United States
  • Z. Wu
    Cole Eye Institute, The Cleveland Clinic Foundation i31, Cleveland, OH, United States
  • Footnotes
    Commercial Relationships  J.W. Crabb, None; A. Hasan, None; Z. Wu, None.
  • Footnotes
    Support  Supported in part by NIH grants EY06603, EY14239, a Research Center Grant from The Foundation Fighti
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3511. doi:
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      J.W. Crabb, A. Hasan, Z. Wu; CRALBP Topological Analyses by Mass Spectrometry . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3511.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Cellular retinaldehyde binding protein (CRALBP) interacts with other proteins as well as with ligand in its role as an acceptor of 11-cis-retinol and substrate carrier in the rod visual cycle. CRALBP topological analyses have been pursued as an approach to identifying functional domains and better understanding visual cycle mechanisms. Methods:Human recombinant CRALBP with a 23 residue His-tag N-terminal fusion sequence was produced in E. coli and purified to apparent homogeneity. Amide hydrogen-deuterium exchange (H-D exchange) and mass spectrometric analyses of pepsin digests of apo- and holo-rCRALBP were used to identifying solvent exposed regions that may interact with other proteins and buried regions that may bind 11-cis-retinoid. Results: Significant localized differences in deuterium incorporation were found between apo- and holo-rCRALBP. Ligand dependent conformational changes were observed in rCRALBP residues 4-22, 80-94 and 282-316 which are more solvent exposed in the holo-protein and in residues 198-212 and 224-243 which are less solvent exposed in the holo-protein. Other regions exhibited less than 5% difference in deuterium incorporation between the apo- and holo-proteins. Conclusions:Binding of 11-cis-retinal induces confromational changes in rCRALBP detectable by H/D exchange mass spectrometry. Separate analyses have confirmed retinoid binding pocket components within buried holo-rCRALBP residues 198-212 and 224-243. The present results provide useful hints regarding structural domains in CRALBP available for functional interactions.

Keywords: retinoids/retinoid binding proteins • protein structure/function • retinal pigment epithelium 
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