Abstract
By using a photoactivatable analog of 11-cis-retinal in rhodopsin, we have previously identified the amino acids Phe-115, Ala-117, Glu-122, Trp-126, Ser-127, and Trp-265 as major sites of cross-linking to the chromophore. To further investigate the amino acids that interact with retinal, we have now used site-directed mutagenesis to replace a variety of amino acids in the membrane-embedded helices in bovine rhodopsin, including those that were indicated by cross-linking studies. The mutant rhodopsin genes were expressed in monkey kidney cells (COS-1) and purified. The mutant proteins were studied for their spectroscopic properties and their ability to activate transducin. Substitution of the two amino acids, Trp-265 and Glu-122 by Tyr, Phe, and Ala and by Gln, Asp and Ala, respectively, resulted in blue-shifted (20-30 nm) chromophore, and substitution of Trp-265 by Ala resulted in marked reduction in the extent of chromophore regeneration. Light-dependent bleaching behavior was significantly altered in Ala-117----Phe, Trp-265----Phe, Ala, and Ala-292----Asp mutants. Transducin activation was reduced in these mutants, in particular Trp-265 mutants, as well as in Glu-122----Gln, Trp-126----Leu (Ala), Pro-267----Ala (Asn, Ser), and Tyr-268----Phe mutants. These findings indicate that Trp-265 is located close to retinal and Glu-122, Trp-126, and probably Tyr-268 are also likely to be near retinal.
Highlights
By using a photoactivatable analog of 11-cis-retinal segment plasma membrane andfinally, results in hyperpolarin rhodopsin, we have previously identified the amino ization of the rod cell plasma membrane generating a neural acids Phe-115, Ala-117, Glu-122, Trp-126, Ser-127, signal ( 5 )
To further investigate the amino acids that interact withretinal, we have used sitedirected mutagenesis to replace a variety ofamino acids in the membrane-embeddedhelices in bovine rhodopsin, including those that were indicated by crosslinking studies
In a previous study [6], we used a photoactivatable analog of 11-cis-retinal to study cross-linking between the retinal and theneighboring amino acids
Summary
296, the site of attachment of retinal, is boxed. Theamino acids substitutedin the present work are circled. Tion fragment (Table I) of the gene in pTN2 was replaced with a synthetic duplex containing H211R, Y268F, Y274F, or A292D mutation, respectively. Construction of Rhodopsin Mutants -Mutations were introduced purified wild-type or mutant rhodopsin and 10 pM [35S]GTP-ySin a into the synthetic opsin gene by restriction fragment replacements buffer containing 10 mM Tris-HC1, pH 7.4, 100 mM NaC1, 5 mM 0-. For the Trp-265 and Pro-267 substitutions, the MluI-NdeI a 40-pl aliquot was rapidlyfiltered through anitrocellulose filter fragment (50/48nucleotides) of the synthetic opsin gene in pOP3 (Schleicher & Schuell, BA 85) and immediately washed three times was replaced with synthetic duplexes (Table I) containing desired with 5 ml of ice-cold buffer. A new vector, pTN2, was constructed toremove redundant restric- the equilibrium assay, the same reaction mixture that contain4sp M tion sites (such as AatII and AhaII) in thevector segment of pOP3. Absorption maxima, the extent of chromophore regeneration, and molar extinction coefficients of rhodomin mutants
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