Disease-causing Mutations in the Cellular Retinaldehyde Binding Protein Tighten and Abolish Ligand Interactions

Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, 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-retinol acceptor and as a substrate carrier. Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, 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-retinol acceptor and as a substrate carrier. cellular retinaldehyde-binding protein autosomal recessive retinitis pigmentosa heteronuclear single quantum correlation liquid chromatography electrospray mass spectrometry retinal pigment epithelium wild type matrix-assisted laser desorption time of flight Mutations in the human gene RLBP1 encoding the cellular retinaldehyde-binding protein (CRALBP)1 cause retinal pathology and have been associated with autosomal recessive retinitis pigmentosa (1Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar), Bothnia dystrophy (2Burstedt M.S.I. Sandreg O. Holmgren G. Forsman-Semb K. Invest. Ophthalmol. Vis. Sci. 1999; 40: 995-1000Google Scholar, 3Burstedt M.S.I. Sandreg O. Forsman-Semb K. Golovleva I. Janunger T. Wachtmeister L. Sandgren O. Arch. Ophthalmol. 2001; 119: 260-267Google Scholar), retinitis punctata albescens (4Morimura H. Berson E.L. Dryja T.P. Invest. Ophthalmol. Vis. Sci. 1999; 40: 1000-1004Google Scholar), fundus albipunctatus (5Katsanis N. Shroyer N.F. Lewis R.A. Cavender J.C. Al-Rajhi A.A. Jabak M. Lupski J.R. Clin. Genet. 2001; 59: 424-429Google Scholar), and Newfoundland rod-cone dystrophy (6Eichers E.R. Green J.S. Stockton D.W. Jackman C. Whelan J. McNamara J.A. Johnson G.J. Lupski J.R. Katsanis N. Am. J. Human Genet. 2002; 70: 955-964Google Scholar). These diseased phenotypes are all characterized by photoreceptor degeneration and night blindness (delayed dark adaptation) but differ in age of onset, rate of progression, and severity. The molecular basis for the clinical differences in these related retinal dystrophies is not well understood and no effective therapies exist for the pathology resulting from impaired CRALBP function. CRALBP is an abundant, 36-kDa protein in the cytosol of the retinal pigment epithelium (RPE) and Müller cells of the retina where it carries endogenous 11-cis-retinol and 11-cis-retinal (7Saari J.C. Sporn M.A. Roberts A.B. Goodman D.S. The Retinoids. Raven Press, Ltd., New York1994: 351-385Google Scholar). The CRALBP ligand binding cavity is mapped in the accompanying report (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar). In vivo studies (9Saari J.C. Nawrot M. Kennedy B.N. Hurley J.B. Garwin G.G. Huang J. Crabb J.W. Neuron. 2001; 29: 739-748Google Scholar) show that CRALBP serves as a major 11-cis-retinol acceptor in the isomerization step of the visual cycle (7Saari J.C. Sporn M.A. Roberts A.B. Goodman D.S. The Retinoids. Raven Press, Ltd., New York1994: 351-385Google Scholar, 10Crouch R.K. Chader G.J. Wiggert B. Pepperberg D.R. Photobiology. 1996; 64: 613-621Google Scholar, 11Rando R.R. Chem. Rev. 2001; 101: 1881-1896Google Scholar), stimulating the enzymatic isomerization of all-trans- to 11-cis-retinol in the rod visual cycle. However, CRALBP appears to function within an RPE protein complex (12Bhattacharya S.K. Wu Z. Jin Z. Yan L. Miyagi M. West K. Nawrot M. Saari J.C. Crabb J.W. FASEB J. 2002; 16: A14Google Scholar) and to serve multiple functions. In vitro, CRALBP facilitates the oxidation of 11-cis-retinol to 11-cis-retinal by 11-cis-retinol dehydrogenase (12Bhattacharya S.K. Wu Z. Jin Z. Yan L. Miyagi M. West K. Nawrot M. Saari J.C. Crabb J.W. FASEB J. 2002; 16: A14Google Scholar, 13Saari J.C. Bredberg D.L. Noy N. Biochemistry. 1994; 33: 3106-3112Google Scholar), retards 11-cis-retinol esterification in the RPE by lecithin:retinol acyltransferase (13Saari J.C. Bredberg D.L. Noy N. Biochemistry. 1994; 33: 3106-3112Google Scholar), and is required for hydrolysis of endogenous RPE 11-cis-retinyl ester (14Stecher H. Gelb M.H. Saari J.C. Palczewski K. J. Biol. Chem. 1999; 274: 8577-8585Google Scholar). Six CRALBP mutations have been linked with retinal pathology, including three missense mutations (R150Q, M225K, and R233W), a frameshift mutation and two predicted splice junction alterations (1Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar, 2Burstedt M.S.I. Sandreg O. Holmgren G. Forsman-Semb K. Invest. Ophthalmol. Vis. Sci. 1999; 40: 995-1000Google Scholar, 4Morimura H. Berson E.L. Dryja T.P. Invest. Ophthalmol. Vis. Sci. 1999; 40: 1000-1004Google Scholar, 6Eichers E.R. Green J.S. Stockton D.W. Jackman C. Whelan J. McNamara J.A. Johnson G.J. Lupski J.R. Katsanis N. Am. J. Human Genet. 2002; 70: 955-964Google Scholar). Recombinant CRALBP (rCRALBP) containing the R150Q mutation lacks the ability to bind 11-cis-retinal and exhibits low solubility (1Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar). Toward a better understanding of the molecular basis of retinal pathology associated with RLBP1gene defects, we report here characterization of rCRALBP containing the M225K or R233W disease-causing mutations (Fig.1). 11-cis-Retinal was obtained from the NEI, National Institutes of Health, and 9-cis-retinal was purchased from Sigma. Tritiated 11-cis-retinol was produced by reduction of 11-cis-retinal with NaB[3H]4 (15Garwin G.G. Saari J.C. Methods Enzymol. 2000; 316: 313-324Google Scholar). Mutant rCRALBP cDNA carrying either the R233W or M225K substitutions were created using The QuikChange site-directed mutagenesis method (Stratagene). Briefly, WT human CRALBP cDNA in the pET19b vector (16Crabb J.W. Carlson A. Chen Y. Goldflam S. Intres R. West K.A. Hulmes J.D. Kapron J.T. Luck L.A. Horwitz J. Bok D. Protein Sci. 1998; 7: 746-757Google Scholar) was cleaved with XbaI andHindIII and the coding region subcloned into pBlueSK. The following complimentary oligonucleotides were used to substitute a Lys for a Met at residue 225 (underlined) in mutant M225K: sense, 5′-GAAGATGGTGGACAAGCTCCAGGATTCCTT-3′; antisense, 5′-AAGGAATCCTGGAGCTTGTCCACCATCTTC-3′. To substitute a Trp for an Arg at residue 233 (underlined) in the R233W mutant, complimentary oligonucleotides were also used: sense, 5′-ATTCCTTCCCAGCCTGGTTCAAAGCCATCC-3′; antisense, 5′-GGATGGCTTTGAACCAGGCTGGGAAGGAAT-3′. Each mutagenesis mix was transformed into Escherichia coli strain XL1-Blue (Stratagene), mutant clones identified by restriction mapping with NspI for M225K and MspI for R233W, were amplified, cleaved with XbaI and HindIII, and ligated back into expression vector pET19b (Novagen). Each insert was sequenced in both directions using the ABI PRISM Dye Terminator Cycle Sequencing kit and the model 377 DNA sequencer (PerkinElmer Life Sciences, Applied Biosystems). WT and rCRALBP mutants M225K and R233W were expressed in E. coli strain BL21(DE3)LysS with a N-terminal His tag and purified using nickel-nitrilotriacetic acid-agarose columns (Qiagen) (16Crabb J.W. Carlson A. Chen Y. Goldflam S. Intres R. West K.A. Hulmes J.D. Kapron J.T. Luck L.A. Horwitz J. Bok D. Protein Sci. 1998; 7: 746-757Google Scholar). Recombinant protein was quantified according to Bradford (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using WT rCRALBP previously quantified by amino acid analysis for the standard reference protein. The masses of the intact mutant proteins were determined by LC ESMS using a PerkinElmer Life Sciences Sciex API 3000 triple quadrupole electrospray mass spectrometer, a Vydac C4 column (1.0 × 150 mm), an Applied Biosystems model 140D high-performance liquid chromatography system and aqueous acetonitrile/trifluoroacetic acid solvents at a flow rate of 50 ॖl/min (18Crabb J.W. Chen Y. Goldflam S. West K. Kapron J. Methods Mole. Biol. 1998; 89: 91-104Google Scholar, 19Crabb J.W. Nie Z. Chen Y. Hulmes J.D. West K.A. Kapron J.T. Ruuska S.E. Noy N. Saari J.C. J. Biol. Chem. 1998; 273: 20712-20720Google Scholar). Phenylthiocarbamyl amino acid analysis was performed with an Applied Biosystems model 420H/130/920 automated system and vapor phase HCl hydrolysis (20Crabb J.W. West K.A. Dodson W.S. Hulmes J.D. Coligan J.E. Ploegh H.L. Smith J.A. Speicher D.W. Current Protocols in Protein Science, Unit 11.9, Supplement 7. John Wiley & Sons, Inc., New York1997: 11.9.1-11.9.42Google Scholar). Purified rCRALBP mutants M225K and R233W (∼100 pmol each) were digested overnight with trypsin, and the peptide digests were analyzed with a PE Biosystems Voyager DE Pro MALDI-TOF mass spectrometer using α-cyano-4-hydroxycinnamic acid as matrix (21West K.A. Yan L. Miyagi M. Crabb J.S. Marmorstein A.D. Marmorstein L. Crabb J.W. Exp. Eye Res. 2001; 73: 479-491Google Scholar, 22Miyagi M. Sakaguchi H. Darrow R.M. Yan L. West K.A. Aulak K.S. Stuehr D.J. Hollyfield J.G. Organisciak D.T. Crabb J.W. Mol. Cell. Proteomics. 2002; 1: 293-303Google Scholar). SDS-PAGE was performed on 107 or 127 acrylamide gels using the Bio-Rad Mini-Protein II system (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar). Retinoid labeling of purified apo-rCRALBP with 11-cis-retinal or 9-cis-retinal, of and analysis by spectroscopy and fluorescence spectroscopy were performed in the as previously (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar, 19Crabb J.W. Nie Z. Chen Y. Hulmes J.D. West K.A. Kapron J.T. Ruuska S.E. Noy N. Saari J.C. J. Biol. Chem. 1998; 273: 20712-20720Google Scholar). Human recombinant 11-cis-retinol dehydrogenase was expressed in cells using a vector by K. Palczewski (12Bhattacharya S.K. Wu Z. Jin Z. Yan L. Miyagi M. West K. Nawrot M. Saari J.C. Crabb J.W. FASEB J. 2002; 16: A14Google Scholar, J.K. Palczewski K. J. Biol. Chem. 2000; Scholar) and purified to apparent by affinity rRDH5 oxidation was at (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar, J.C. Bredberg D.L. Garwin G.G. J. T. Palczewski K. Anal. Biochem. Scholar) and reduction was at S. J. Biol. Chem. Scholar) using purified mutant or WT rCRALBP or amounts of 11-cis-retinol or 11-cis-retinal as substrate (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar). with retinoid as substrate were in the of carrier protein. WT and mutant R233W rCRALBP were by in E. BL21(DE3)LysS in (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar, L.A. R.A. Kapron J. Roth K.A. Crabb J.W. in Protein Press, Scholar). Purified mutant and WT rCRALBP with bound 11-cis-retinal were to and to of NMR Inc., (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar). NMR were performed at with a spectrometer with a triple heteronuclear single quantum correlation were using for was on a using and (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar, S. G. J. A. J. Scholar, D.S. R. J. Scholar). were in the dark or to retinoid WT and mutant were produced in and SDS-PAGE of the soluble and that the M225K mutant was less soluble than the R233W rCRALBP mutant The R233W mutant was in the soluble in amounts comparable to that of the WT protein The purified mutant proteins were characterized by amino acid analysis and by LC ESMS and the determined and intact masses to in with the of mutant protein was by MALDI-TOF peptide mass including the containing the M225K and R233W substitutions acid of human rCRALBP analyzed were determined by amino acid analysis of the purified mutant proteins J.W. Nie Z. Chen Y. Hulmes J.D. West K.A. Kapron J.T. Ruuska S.E. Noy N. Saari J.C. J. Biol. Chem. 1998; 273: 20712-20720Google Scholar). are for or from and Trp were not in a were determined by amino acid analysis of the purified mutant proteins J.W. Nie Z. Chen Y. Hulmes J.D. West K.A. Kapron J.T. Ruuska S.E. Noy N. Saari J.C. J. Biol. Chem. 1998; 273: 20712-20720Google Scholar). are for or from and Trp were not UV-visible analysis of purified mutant R233W and WT rCRALBP with bound 11-cis-retinal were with bound 9-cis-retinal the ligand is for the R233W mutant to the for both to to the of In contrast, UV-visible of purified mutant M225K no for binding of either or 9-cis-retinal Retinoid labeling performed in to protein the UV-visible results not for mutant R233W rCRALBP with 11-cis- or 9-cis-retinal were determined by fluorescence of the the in the fluorescence of the protein upon ligand binding The significantly affinity of the R233W mutant for both 11-cis-retinal and 9-cis-retinal relative to WT rCRALBP for 11-cis-retinal and for The of binding from the mutant R233W was for either of rCRALBP with S.E. are in a S.E. are rRDH5 was assayed in the of the M225K rCRALBP mutant were obtained relative to WT rCRALBP for both oxidation and reduction rRDH5 was assayed using retinoid as determined were to higher than WT rCRALBP to the retinoid mutation to significantly In vivo CRALBP is to the oxidation of 11-cis-retinol to 11-cis-retinal (13Saari J.C. Bredberg D.L. Noy N. Biochemistry. 1994; 33: 3106-3112Google Scholar) and in for WT rCRALBP was in the oxidation relative to 11-cis-retinol and than the was using WT rCRALBP or 11-cis-retinal in the reduction The results impaired substrate carrier function in the oxidation for the M225K and R233W rCRALBP of mutant CRALBP substrate carrier dehydrogenase of 11-cis-retinal to 11-cis-retinol Mutant R233W Mutant M225K WT CRALBP 11-cis-retinal of 11-cis-retinol to 11-cis-retinal Mutant R233W Mutant M225K WT CRALBP 11-cis-retinol reduction and oxidation were with purified recombinant 11-cis-retinol dehydrogenase in the or of the purified mutant and wild type rCRALBP as are for in a reduction and oxidation were with purified recombinant 11-cis-retinol dehydrogenase in the or of the purified mutant and wild type rCRALBP as are for The heteronuclear single quantum correlation NMR for rCRALBP and WT were in the dark and for for and in NMR of WT rCRALBP were in the accompanying report (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar). have also been for the Met in WT rCRALBP (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar) and and Mutant R233W a of three and was in on the in the region of Trp differences in structures WT rCRALBP and mutant R233W are demonstrated by the WT and R233W that not as differences within the retinoid binding cavity are by an apparent retinoid binding pocket (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar), not with in the R233W In NMR were and of rCRALBP to The results show that the of by ligand changes are apparent in the R233W upon retinoid but the changes are less extensive than upon the WT protein single quantum correlation NMR of mutant R233W and WT rCRALBP and The correlation for the proteins with bound 11-cis-retinal was in the dark and the was to and by the correlation mutant R233W The of the in both upon ligand isomerization in the R233W ligand binding WT rCRALBP upon (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar), more changes are apparent than in the R233W impaired CRALBP was associated with autosomal recessive retinitis pigmentosa in the missense mutation R150Q was in the RLBP1 gene from a in (1Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar). pigmentosa is a of with and and of the disease to J.K. Bok D. Mol. Vis. 2000; Scholar). recessive in gene have been to cause retinal including the two missense mutations M225K and R233W associated with retinitis punctata albescens and Bothnia dystrophy (2Burstedt M.S.I. Sandreg O. Holmgren G. Forsman-Semb K. Invest. Ophthalmol. Vis. Sci. 1999; 40: 995-1000Google Scholar, N. Shroyer N.F. Lewis R.A. Cavender J.C. Al-Rajhi A.A. Jabak M. Lupski J.R. Clin. Genet. 2001; 59: 424-429Google Scholar). mutations in the CRALBP gene have been associated with phenotypes and in from the and (1Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar, 2Burstedt M.S.I. Sandreg O. Holmgren G. Forsman-Semb K. Invest. Ophthalmol. Vis. Sci. 1999; 40: 995-1000Google Scholar, 3Burstedt M.S.I. Sandreg O. Forsman-Semb K. Golovleva I. Janunger T. Wachtmeister L. Sandgren O. Arch. Ophthalmol. 2001; 119: 260-267Google Scholar, 4Morimura H. Berson E.L. Dryja T.P. Invest. Ophthalmol. Vis. Sci. 1999; 40: 1000-1004Google Scholar, N. Shroyer N.F. Lewis R.A. Cavender J.C. Al-Rajhi A.A. Jabak M. Lupski J.R. Clin. Genet. 2001; 59: 424-429Google Scholar, 6Eichers E.R. Green J.S. Stockton D.W. Jackman C. Whelan J. McNamara J.A. Johnson G.J. Lupski J.R. Katsanis N. Am. J. Human Genet. 2002; 70: 955-964Google Scholar). RLBP1 gene are to a cause of retinal disease (4Morimura H. Berson E.L. Dryja T.P. Invest. Ophthalmol. Vis. Sci. 1999; 40: 1000-1004Google in the of Bothnia dystrophy by the R233W mutation a for therapies are (2Burstedt M.S.I. Sandreg O. Holmgren G. Forsman-Semb K. Invest. Ophthalmol. Vis. Sci. 1999; 40: 995-1000Google Scholar, 3Burstedt M.S.I. Sandreg O. Forsman-Semb K. Golovleva I. Janunger T. Wachtmeister L. Sandgren O. Arch. Ophthalmol. 2001; 119: 260-267Google Scholar). To better understand the molecular basis of retinal pathology associated with impaired CRALBP and for we have studies of the mutant containing the disease causing substitutions M225K and The structural of the purified M225K and R233W mutant recombinant proteins was by amino acid analysis and mass In to the R150Q rCRALBP associated with (1Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar), the R233W mutant exhibits solubility comparable to that of WT rCRALBP, the M225K mutant is less soluble than WT rCRALBP but significantly more soluble than the R150Q to retinoid binding UV-visible analysis that the M225K mutant the R150Q mutant and lacked the ability to bind (1Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar). In contrast, mutant R233W bound stoichiometric amounts of 11-cis- or 9-cis-retinal on J.W. Nie Z. Chen Y. Hulmes J.D. West K.A. Kapron J.T. Ruuska S.E. Noy N. Saari J.C. J. Biol. Chem. 1998; 273: 20712-20720Google Scholar). apparent for the R233W mutant that demonstrated for and 11-cis-retinal that were 2-fold than determined for WT rCRALBP. The of the retinoid affinity standard of the was was within the of of the and in to the low aqueous solubility of and the protein used in the fluorescence was significantly higher than the these to for the N. H. W.S. of Scholar). The the binding of WT rCRALBP and R233W mutant than with from RPE (13Saari J.C. Bredberg D.L. Noy N. Biochemistry. 1994; 33: 3106-3112Google Scholar), and purified proteins (12Bhattacharya S.K. Wu Z. Jin Z. Yan L. Miyagi M. West K. Nawrot M. Saari J.C. Crabb J.W. FASEB J. 2002; 16: A14Google Scholar) a substrate carrier CRALBP and The performed here with purified recombinant proteins that rCRALBP either the M225K or the R233W mutations the apparent affinity of rRDH5 for 11-cis-retinol and 11-cis-retinal relative to the WT protein. The of rRDH5 for 11-cis-retinoid in the of the M225K mutant that for the with retinoid (12Bhattacharya S.K. Wu Z. Jin Z. Yan L. Miyagi M. West K. Nawrot M. Saari J.C. Crabb J.W. FASEB J. 2002; 16: A14Google Scholar). higher was obtained for rRDH5 using the mutant as affinity and retinoid to the binding of retinoid by The R233W mutant of the substrate to the of These are with the that CRALBP the of by of to the cellular acid binding protein II facilitates of acid to the acid D. Ruuska S. D.J. Noy N. J. Biol. Chem. 1999; 274: Scholar, R. Noy N. J. Mol. Biol. 2001; Scholar). to the structural basis for the retinoid binding properties of the R233W mutant were obtained by NMR that the structure of the R233W mutant with bound ligand was significantly different than that of WT including different retinoid-binding cavity changes by NMR photoisomerization of 11-cis-retinal in the ligand binding pocket were more for the WT protein (8Wu Z. Yang Y. Shaw N. Bhattacharya S. Yan L. West K. Roth K. Noy N. Qin J. Crabb J.W. J. Biol. Chem. 2003; 278: 12390-12396Google Scholar) than for the R233W mutant and are within and CRALBP and the results are with ligand with both with within the ligand binding resulting in a more less R233W protein in of appears to within the retinoid binding by the region to in ligand and M225K protein The function of CRALBP in the visual cycle upon the and of retinoid from the ligand binding The results of of both retinoid binding and as of the night blindness and retinal pathology for human with CRALBP CRALBP serves as the major 11-cis-retinol acceptor in the isomerization of the rod visual the of retinoid binding by M225K rCRALBP significantly the enzymatic of to as for the CRALBP (9Saari J.C. Nawrot M. Kennedy B.N. Hurley J.B. Garwin G.G. Huang J. Crabb J.W. Neuron. 2001; 29: 739-748Google Scholar). of 11-cis-retinol by with oxidation to 11-cis-retinal by a for isomerization (13Saari J.C. Bredberg D.L. Noy N. Biochemistry. 1994; 33: 3106-3112Google Scholar, J.K. R. O. I. Palczewski K. Biochemistry. 2000; Scholar). with proteins the of ligand from the CRALBP binding than stoichiometric retinoid binding (9Saari J.C. Nawrot M. Kennedy B.N. Hurley J.B. Garwin G.G. Huang J. Crabb J.W. Neuron. 2001; 29: 739-748Google S.K. Wu Z. Jin Z. Yan L. Miyagi M. West K. Nawrot M. Saari J.C. Crabb J.W. FASEB J. 2002; 16: A14Google Scholar). The R233W mutation results in rCRALBP retinoid binding and rRDH5 affinity for rCRALBP bound The retinoid binding by the R233W mutation appears to the of ligand, resulting in a that 11-cis-retinol acceptor function and the isomerization of to John C. Saari for and for the to with

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