title: Microsecond time-resolved circular dichroism of rhodopsin photointermediates.
abstract: Time-resolved circular dichroism measurements, over a spectral range from 300 to 700 nm, were made at delays of 5, 100, and 500 micros after room-temperature photoexcitation of bovine rhodopsin in a lauryl maltoside suspension. The purpose was to provide more structural information about intermediate states in the activation of rhodopsin and other G protein-coupled receptors. In particular, information was sought about photointermediates that are isochromic or nearly isochromic in their unpolarized absorbance. The circular dichroism spectrum of lumirhodopsin, obtained after correcting the 5 micros difference CD data for the bleached rhodopsin, was in reasonable agreement with the lumirhodopsin CD spectrum obtained previously by thermal trapping at -76 degrees C. Similarly, the metarhodopsin II spectrum obtained with a 500 micros delay was also in agreement with the results of previous work on the temperature-trapped form of metarhodopsin II. However, the CD of the mixture formed with a 100 micros delay after photoexcitation, whose only visible absorbing component is lumirhodopsin, could not be accounted for near 480 nm in terms of the initially formed, 5 micros lumirhodopsin CD spectrum. Thus, the CD spectrum of lumirhodopsin changes on the time scale from 5 to 100 micros, showing reduced rotational strength in its visible band, possibly associated with either a process responsible for a small spectral shift that occurs in the lumirhodopsin absorbance spectrum at earlier times or the Schiff base deprotonation-reprotonation which occurs during equilibration of lumirhodopsin with the Meta I(380) photointermediate. Either explanation suggests a chromophore conformation change closely associated with deprotonation which could be the earliest direct trigger of activation.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: Yiren Gu
authors.initials: YG
authors.lastname: Thomas
authors.forename: Istvan
authors.initials: I
authors.lastname: Szundi
authors.forename: James W
authors.initials: JW
authors.lastname: Lewis
authors.forename: David S
authors.initials: DS
authors.lastname: Kliger
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism / Q000379:methods*; D007700:Kinetics; D055668:Photochemical Processes; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry*
chemical_list: D012243:Rhodopsin