title: Solid-state 13C NMR of the retinal chromophore in photointermediates of bacteriorhodopsin: characterization of two forms of M.
abstract: Solid-state 13C NMR spectra of the M photocycle intermediate of bacteriorhodopsin (bR) have been obtained from purple membrane regenerated with retinal specifically 13C labeled at positions 5, 12, 13, 14, and 15. The M intermediate was trapped at -40 degrees C and pH = 9.5-10.0 in either 100 mM NaCl [M (NaCl)] or 500 mM guanidine hydrochloride [M (Gdn-HCl)]. The 13C-12 chemical shift at 125.8 ppm in M (NaCl) and 128.1 ppm in M (Gdn-HCl) indicates that the C13 = C14 double bond has a cis configuration, while the 13C-13 chemical shift at 146.7 ppm in M (NaCl) and 145.7 ppm in M (Gdn-HCl) demonstrates that the Schiff base is unprotonated. The principal values of the chemical shift tensor of the 13C-5 resonance in both M (NaCl) and M (Gdn-HCl) are consistent with a 6-s-trans structure and a negative protein charge localized near C-5 as was observed in dark-adapted bR. The approximately 5 ppm upfield shift of the 13C-5 M resonance (approximately 140 ppm) relative to 13C-5 bR568 and bR548 (approximately 145 ppm) is attributed to an unprotonated Schiff base in the M chromophore. Of particular interest in this study were the results obtained from 13C-14 M. In M (NaCl), a dramatic upfield shift was observed for the 13C-14 resonance (115.2 ppm) relative to unprotonated Schiff base model compounds (approximately 128 ppm). In contrast, in M (Gdn-HCl) the 13C-14 resonance was observed at 125.7 ppm. The different 13C-14 chemical shifts in these two M preparations may be explained by different C = N configurations of the retinal-lysine Schiff base linkage, namely, syn in NaCl and anti in guanidine hydrochloride.
authors.affiliation: Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology 02139.
authors.forename: S O
authors.initials: SO
authors.lastname: Smith
authors.forename: J
authors.initials: J
authors.lastname: Courtin
authors.forename: E
authors.initials: E
authors.lastname: van den Berg
authors.forename: C
authors.initials: C
authors.lastname: Winkel
authors.forename: J
authors.initials: J
authors.lastname: Lugtenburg
authors.forename: J
authors.initials: J
authors.lastname: Herzfeld
authors.forename: R G
authors.initials: RG
authors.lastname: Griffin
mesh_terms: D001436:Bacteriorhodopsins; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010777:Photochemistry; D011487:Protein Conformation; D012172:Retinaldehyde
chemical_list: D001436:Bacteriorhodopsins; D012172:Retinaldehyde