| The cellular retinaldehyde-binding protein (CRALBP) serves in the rod visual cycle as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). To identify amino acids in the CRALBP retinoid-binding cavity, human recombinant CRALBP Met and Trp mutants were produced and analyzed, including M208A, M222A, M225A, W 165F and W244F. Hydrogen deuterium exchange and photoaffinity labeling analyses were also performed. All Met and Tip rCRALBP mutants bound 11-cis- and 9-cis-retinal with altered UV-visible spectra and lower retinoid binding affinities. rCRALBP mutants M222A, M225A and W244F exhibited altered kinetics and impaired substrate carrier function for 11-cis-retinol dehydrogenase. Heteronuclear single quantum correlation (HSQC) NMR confirmed localized structural changes upon photoisomerization of wildtype rCRALBP complexed with 11-cis-retinal and identified ligand-dependent conformational changes in mutant rCRALBPs, including residues M208, M222, W165 and W244. Photoaffinity labeling identified residues Y179, F197, C198, M 208, K221, M222, V223 and M225 as components of the CRALBP retinoid-binding pocket. H/D exchange and mass spectrometry demonstrated rCRALBP residues 197--244 exhibit ligand dependent solvent accessibility. This region contains ten of the twelve identified ligand binding pocket residues. rCRALBP mutants M225K and R233W were also produced and analyzed to understand the molecular basis of human blindness associated with these mutations. The M225K mutant lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant bound 11-cis and 9-cis-retinal with higher affinity than wildtype rCRALBP and decreased the apparent affinity of rRDH5 for 11-cis-retinoid. HSQC NMR demonstrated that mutant R233W exhibits a different conformation than WT rCRALBP. Overall, the thesis results support W165, Y179, F197, C198, M208, K221, M222, V223, M225, 8233 and W244 as components of the CRALBP retinoid-binding cavity. Furthermore, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinal acceptor and as a substrate carrier. |
