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S.K. Bhattacharya, Z. Wu, K. Nakanishi, J.W. Crabb; Identification of the CRALBP Ligand Binding Pocket by Photoaffinity Labeling . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3512.
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Purpose: Noncovalent ligand interactions determine the function of cellular retinaldehyde-binding protein (CRALBP) in the rod visual cycle as an 11-cis-retinol acceptor and substrate carrier. To further characterize the ligand binding cavity of CRALBP, we covalently labeled rCRALBP with a photoaffinity retinoid analogue and identified the modified amino acids. Methods: Purified human apo-rCRALBP was labeled with 3-diazo-4-keto-11-cis-retinal, excess retinoid removed and covalent incorporation achieved by photolysis with UV-light (254 nm) at –1960C for 5s to 20 min. Protein bound retinal was reduced to retinol with NaB3H4, radiolabeling the ligand incorporation sites. Labeled rCRALBP was alkylated, digested with trypsin and fractionated by RP-HPLC. Radioactive peptide fractions were identified and peptides were analyzed by MALDI-TOF MS and LC MS/MS. Results: Short irradiation times (5-40s) yielded relatively constant incorporation levels (~1%) therefore 5s photolysis times were used to minimize nonspecific protein modifications. Peptides accounting for 100% of the rCRALBP sequence were identified by mass spectrometric analyses of photolabeled rCRALBP. Eight photoaffinity modified residues were identified in radioactive fractions, each with variable mass additions. Conclusions: Four of the photoaffinity modified sites have been previously identified as retinoid binding pocket residues. The other four modified residues are also in the retinoid binding domain and may represent newly identified CRALBP ligand binding pocket components. The cause of the variable mass additions is not clear but may be due in part to free radical migration throughout the conjugated double bonds of the retinoid analogue.
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