Microsecond time-resolved circular dichroism of rhodopsin photointermediates.
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.
Thomas
Yiren Gu
YG
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Szundi
Istvan
I
Lewis
James W
JW
Kliger
David S
DS
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism / Q000379:methods*; D007700:Kinetics; D055668:Photochemical Processes; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry*
D012243:Rhodopsin