title: Vibrational spectroscopy of bacteriorhodopsin mutants: evidence for the interaction of proline-186 with the retinylidene chromophore.
abstract: 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.
authors.affiliation: Physics Department, Boston University, Massachusetts 02215.
authors.forename: K J
authors.initials: KJ
authors.lastname: Rothschild
authors.forename: Y W
authors.initials: YW
authors.lastname: He
authors.forename: T
authors.initials: T
authors.lastname: Mogi
authors.forename: T
authors.initials: T
authors.lastname: Marti
authors.forename: L J
authors.initials: LJ
authors.lastname: Stern
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: 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
chemical_list: D011522:Protons; D012168:Retinal Pigments; D012545:Schiff Bases; D014443:Tyrosine; D001436:Bacteriorhodopsins; D001216:Asparagine; D011392:Proline; D014633:Valine