Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin. The simpler case of the recombinant D96N protein

摘要: The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of their interconversions has precluded a rigorous solution kinetics. A representation mutated D96N near neutral pH was given earlier (Varo, G., J. K. Lanyi. 1991. Biochemistry. 30:5008–5015) as BRhv-->K L M1-->M2--> BR. Here we have reduced set time-resolved difference spectra for this simpler system to three base spectra, each assumed consist an unknown mixture pure K, L, M represented by 3 x matrix concentration values between 0 1. After generating all allowed sets (i.e., M1 + M2) at 1:50 resolution elements, invalid solutions were eliminated progressively in search based on what is expected, empirically from theory polyene excited states, rhodopsin spectra. Significantly, average changed little after first simplest criteria that disallowed negative absorptions one maximum intermediate. We conclude statistics during strongly converged into narrow region multidimensional space matrix. data temperatures 5 25 degrees C yielded single M; fits are consistent with derived model protein.