title: A mutant rhodopsin photoproduct with a protonated Schiff base displays an active-state conformation: a Fourier-transform infrared spectroscopy study.
abstract: In the rhodopsin mutant E113A/A117E the position of the protonated Schiff base counterion, Glu113, is moved by one helix turn from position 113 to 117. The photoreaction of this mutant pigment was studied by Fourier-transform infrared (FTIR) difference spectroscopy. At acidic pH, formation of a 474-nm absorbing photoproduct previously characterized biochemically as a species that activates transducin caused infrared absorption changes typical of metarhodopsin II (MII) formation in native rhodopsin. Specific spectral alterations revealed a localized perturbation near the protonated Schiff base in the dark state. In addition, an infrared band assigned to the C = O stretching vibration of Glu113 in MII of rhodopsin was abolished in the mutant. Absorption changes caused by Asp83 and Glu122 C = O stretching vibrations characteristic of rhodopsin MII formation were not affected. At alkaline pH, mutant E113A/A117E formed predominantly a 382-nm absorbing photoproduct. It displayed infrared-difference absorption bands significantly different from those of native MII over a large spectral range. These results support the conclusion that the 474-nm photoproduct of mutant E113A/A117E, despite a protonated Schiff base linkage, displays a predominantly MII-like conformation capable of catalyzing guanine-nucleotide exchange by transducin.
authors.affiliation: Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
authors.forename: K
authors.initials: K
authors.lastname: Fahmy
authors.forename: F
authors.initials: F
authors.lastname: Siebert
authors.forename: T P
authors.initials: TP
authors.lastname: Sakmar
mesh_terms: D002264:Carboxylic Acids / Q000737:chemistry; D006860:Hydrogen Bonding; D009154:Mutation; D010777:Photochemistry; D011487:Protein Conformation; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D002264:Carboxylic Acids; D011522:Protons; D012545:Schiff Bases; D012243:Rhodopsin