Structure-function studies on bacteriorhodopsin. VIII. Substitutions of the membrane-embedded prolines 50, 91, and 186: the effects are determined by the substituting amino acids.
To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded alpha-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50----Ala, Pro-91----Ala and ----Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50----Gly was 10-fold faster than in the wild-type. While, Pro-186----Ala regenerated the wild-type chromophore, the mutants Pro-186----Val and Pro-186----Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186----Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentration restored the wild-type purple chromophore in the Pro-186----Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186----Ala and Pro-186----Gly showed about 65%, whereas Pro-186----Val showed 10-20% of the normal proton pumping.
Mogi
T
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Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Stern
L J
LJ
Chao
B H
BH
Khorana
H G
HG
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D005998:Glycine / Q000235:genetics; D008027:Light; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D011522:Protons; D012965:Sodium Chloride; D013329:Structure-Activity Relationship; D014633:Valine / Q000235:genetics
D008565:Membrane Proteins; D011522:Protons; D012965:Sodium Chloride; D001436:Bacteriorhodopsins; D011392:Proline; D014633:Valine; D000409:Alanine; D005998:Glycine