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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)
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.
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