title: Complete identification of C = O stretching vibrational bands of protonated aspartic acid residues in the difference infrared spectra of M and N intermediates versus bacteriorhodopsin.
abstract: Fourier transform infrared difference spectra were obtained for the M and N intermediates versus light-adapted bacteriorhodopsin (BR) with site-directed mutant proteins in which aspartic acid residues at positions 96 and 115 were replaced by asparagine. The positive and negative bands at 1740 and 1732 cm-1 in the M/BR spectrum are shown to be the superposition of bands due to C = O stretching vibrations of Asp-96 and Asp-115 (a positive band at 1736 cm-1 and a negative band at 1742 cm-1 of Asp-96, and a positive band at 1742 cm-1 and a negative band at 1734 cm-1 of Asp-115). The positive band at 1738 cm-1 and the negative band at 1734 cm-1 in the N/BR spectrum are attributed to Asp-115. On the basis of these results, Asp-115 is protonated in M and N as well as in the ground state. On the other hand, no bands corresponding to Asp-212 were found in the region of protonated carboxylic acid vibration, indicating that Asp-212 remains unprotonated in M and N. The frequencies of the C = O stretching modes of protonated Asp-96 and Asp-115 change in the opposite direction in the BR-to-M conversion relative to the shifts in the BR-to-L conversion, indicating different environmental changes for these residues in L and M.
authors.affiliation: Department of Biophysics, Faculty of Science, Kyoto University, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Sasaki
authors.forename: J K
authors.initials: JK
authors.lastname: Lanyi
authors.forename: R
authors.initials: R
authors.lastname: Needleman
authors.forename: T
authors.initials: T
authors.lastname: Yoshizawa
authors.forename: A
authors.initials: A
authors.lastname: Maeda
mesh_terms: D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D002244:Carbon / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry*; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D011522:Protons; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine; D002244:Carbon; D010100:Oxygen