Microsecond Time-Resolved Circular Dichroism of Rhodopsin Photointermediates

作者：Thomas Yiren Gu; Szundi Istvan; Lewis James W; Kliger David S^*

来源：Biochemistry, 2009, 48(51): 12283-12289.

DOI：10.1021/bi901657b

Time-resolved circular dichroism measurements, over a spectral range from 300 to 700 nm, were made at delays of 5, 100, and 500 mu s 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 mu s 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 mu s 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 mu s 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 mu s lumirhodopsin CD spectrum. Thus, the CD spectrum of lumirhodopsin changes on the time scale from 5 to 100 mu s, 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-reproton 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.

• 出版日期2009-12-29