Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin

The simpler case of the recombinant D96N protein

L. Zimányi, J. K. Lanyi

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Abstract

The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of the interconversions has precluded a rigorous solution of the kinetics. A representation of the photocycle of mutated D96N bacteriorhodopsin near neutral pH was given earlier (Varo, G., and J. K. Lanyi. 1991. Biochemistry. 30:5008-5015) as BR→(hv)→K⇆L⇆M1→M2→BR. Here we have reduced a set of time-resolved difference spectra for this simpler system to three base spectra, each assumed to consist of an unknown mixture of the pure K, L, and M difference spectra represented by a 3x3 matrix of concentration values between 0 and 1. After generating all allowed sets of spectra for K, L, and M (i.e., M1 + M2) at a 1:50 resolution of the matrix elements, invalid solutions were eliminated progressively in a search based on what is expected, empirically and from the theory of polyene excited states, for rhodopsin spectra. Significantly, the average matrix values changed little after the first and simplest of the search criteria that disallowed negative absorptions and more than one maximum for the M intermediate. We conclude from the statistics that during the search the solutions strongly converged into a narrow region of the multidimensional space of the concentration matrix. The data at three temperatures between 5 and 25°C yielded a single set of spectra for K, L, and M; their fits are consistent with the earlier derived photocycle model for the D96N protein.

Original languageEnglish
Pages (from-to)240-251
Number of pages12
JournalBiophysical Journal
Volume64
Issue number1
Publication statusPublished - 1993

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Bacteriorhodopsins
Recombinant Proteins
Polyenes
Rhodopsin
Biochemistry
Temperature
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

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title = "Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin: The simpler case of the recombinant D96N protein",
abstract = "The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of the interconversions has precluded a rigorous solution of the kinetics. A representation of the photocycle of mutated D96N bacteriorhodopsin near neutral pH was given earlier (Varo, G., and J. K. Lanyi. 1991. Biochemistry. 30:5008-5015) as BR→(hv)→K⇆L⇆M1→M2→BR. Here we have reduced a set of time-resolved difference spectra for this simpler system to three base spectra, each assumed to consist of an unknown mixture of the pure K, L, and M difference spectra represented by a 3x3 matrix of concentration values between 0 and 1. After generating all allowed sets of spectra for K, L, and M (i.e., M1 + M2) at a 1:50 resolution of the matrix elements, invalid solutions were eliminated progressively in a search based on what is expected, empirically and from the theory of polyene excited states, for rhodopsin spectra. Significantly, the average matrix values changed little after the first and simplest of the search criteria that disallowed negative absorptions and more than one maximum for the M intermediate. We conclude from the statistics that during the search the solutions strongly converged into a narrow region of the multidimensional space of the concentration matrix. The data at three temperatures between 5 and 25°C yielded a single set of spectra for K, L, and M; their fits are consistent with the earlier derived photocycle model for the D96N protein.",
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