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Structure-Function Studies of Bacteriorhodopsin XV



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Vol 266 No 13 Issue of May 5 pp 8545 8550 1991 Printed in U S A THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology Inc Structure Function Studies of BacteriorhodopsinXV EFFECTSOFDELETIONSIN ANDSTRUCTURE LOOPS B CANDE FONBACTERIORHODOPSINCHROMOPHORE Received for publication November 15 1990 Marie A Gilles GonzalezSQ Donald M Engelmann and H Gobind KhoranaS From the Departments of Biology and Chemistry Massachusetts Institute of Technology Cambridge Massachusetts 02139 and the TDepartment of Molecular Biophysics and Biochemistry Yale university New Haven Connecticut 06511 various modelsproposed 6 9 andphotoaffinity labeling experiments have indicated that E F is also a large loop 7 In the present work we examine theeffects of deletions in the loops on the folding function and stability of bR Deletions of amino acids T h r j G l a n d G l j G in l n the loop connecting helices B and C were made andof amino acids G1ulfi1AlaIfi8in the loop between helices E and F The B C loop lies on the extracellular side of the membrane and the E F loop on the cytoplasmicside We found that all the deletions destabilize the structure to some extent as measuredby sensitivity to thermal and SDS denaturation No effect was seen on proton pumping and the absorption spectrum in the caseof the E Fdeletion however the two deletions in loop B C show blue shifts in the chromophore absorption maximum diminished proton pumping and increased susceptibility to hydroxylamine bleaching In all cases the basic folding of bR occurs as judged by circular dichroism chromophore binding and protonpumping EXPERIMENTALPROCEDURES Bacteriorhodopsin bR l an integral membrane proteinin Halobacterium halobium serves as a light dependent proton pump 1 It consists of a single polypeptide chain of 248 amino acids and contains the chromophore all tram retinal linked via a Schiff base tolysine 216 2 5 The model shown in Fig 1 is based mainly on electronmicroscopy with



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