Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F.
1987; Elsevier BV; Volume: 262; Issue: 19 Linguagem: Inglês
10.1016/s0021-9258(18)48077-5
ISSN1083-351X
AutoresNeil R. Hackett, Lawrence J. Stern, B H Chao, K. Anne Kronis, H. G. Khorana,
Tópico(s)Origins and Evolution of Life
ResumoTo test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied.Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore.Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli.The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/ detergent micelles to form a bacteriorhodopsin-like chromophore.Four mutants, Ser-183 + Ala, Tyr-185 + Phe, Ser-193 + Ala, and Glu-194 + Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type.Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type.Two Trp + Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186 + Leu gave a pigment with an absorption maximum of 470 nm.However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.Substitution of specific amino acids in proteins provides an attractive and versatile approach to their structure-function studies.Such an approach is made possible by the techniques of recombinant DNA and is being currently used in studies of a number of proteins (1).Bacteriorhodopsin (bR),' an integral membrane protein from Halobacterium hulobium, carries out light-dependent proton translocation from the inside to the
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