Vibrational spectroscopy of bacteriorhodopsin mutants: evidence for the interaction of proline-186 with the retinylidene chromophore.
Fourier-transform infrared difference spectroscopy has been used to study the role of the three membrane-embedded proline residues, Pro-50, Pro-91, and Pro-186, in the structure and function of bacteriorhodopsin. All three prolines were replaced by alanine and glycine; in addition, Pro-186 was changed to valine. Difference spectra were recorded for the bR----K and bR----M photoreactions of each of these mutants and compared to those of wild-type bacteriorhodopsin. Only substitutions of Pro-186 caused significant perturbations in the frequency of the C = C and C - C stretching modes of the retinylidene chromophore. In addition, these substitutions reduced bands in the amide I and II region associated with secondary structural changes and altered signals assigned to the adjacent Tyr-185. Pro-186----Val caused the largest alterations, producing a second species similar to bR548 and nearly blocking chromophore isomerization at 78 K but not at 250 K. These results are consistent with a model of the retinal binding site in which Pro-186 and Tyr-185 are located in direct proximity to the chromophore and may be involved in linking chromophore isomerization to protein structural changes. Evidence is also found that Pro-50 may be structurally active during the bR----K transition and that substitution of this residue by glycine preserves the normal protein structural changes during the photocycle.
Rothschild
K J
KJ
Physics Department, Boston University, Massachusetts 02215.
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Y W
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Mogi
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Marti
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Stern
L J
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Khorana
H G
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D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001436:Bacteriorhodopsins / Q000235:genetics* / Q000378:metabolism; D005583:Fourier Analysis; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons; D012168:Retinal Pigments / Q000378:metabolism; D012545:Schiff Bases / Q000378:metabolism; D013055:Spectrophotometry, Infrared / Q000379:methods; D013057:Spectrum Analysis / Q000379:methods; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000235:genetics; D014732:Vibration
D011522:Protons; D012168:Retinal Pigments; D012545:Schiff Bases; D014443:Tyrosine; D001436:Bacteriorhodopsins; D001216:Asparagine; D011392:Proline; D014633:Valine