Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer

Kakareka, John W.; Smith, Paul D.; Pohida, Thomas J.; Hendler, Richard W.

doi:10.1016/j.jprot.2007.11.004

Cited by 2 publications

(1 citation statement)

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“…For a complete description of the spectrometers used and the visible optics, see reference 4 . The basic system uses two separate spectrographs, one covering a range of about 390 nm to 521 nm and another from about 553 nm to 681 nm with a resolution of 2.9 nm.…”

Section: Methodsmentioning

confidence: 99%

Infrared and Visible Absolute and Difference Spectra of Bacteriorhodopsin Photocycle Intermediates

Hendler

Meuse

Braiman³

et al. 2011

Appl Spectrosc

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We have used new kinetic fitting procedures to obtain IR absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The linear algebra-based procedures of Hendler et al. (2001) J. Phys. Chem. B, 105, 3319–3228, for obtaining clean absolute visible spectra of bR photocycle intermediates, were adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra, including the separation of the M intermediate into its MF and MS components from parallel photocycles. This in turn permitted the computation of clean IR difference spectra between pairs of successive intermediates, allowing for the most rigorous analysis to date of changes occurring at each step of the photocycle. The statistical accuracy of the spectral calculation methods allows us to identify, with great confidence, new spectral features. One of these is a very strong differential IR band at 1650 cm−1 for the L intermediate at room temperature that is not present in analogous L spectra measured at cryogenic temperatures. This band, in one of the noisiest spectral regions, has not been identified in any previous time-resolved IR papers, although retrospectively it is apparent as one of the strongest L absorbance changes in their raw data, considered collectively. Additionally, our results are most consistent with Arg82 as the primary proton-release group (PRG), rather than a protonated water cluster or H-bonded grouping of carboxylic residues. Notably, the Arg82 deprotonation occurs exclusively in the MF pathway of the parallel cycles model of the photocycle.

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Section: Methodsmentioning

confidence: 99%