Infrared and Visual Absolute and Difference Spectra of Bacteriorhodopsin Photocycle Intermediates Part 1: Isolation of Spectra
Curtis W. Meuse, R W. Hendler, P D. Smith, John Kakareka
New procedures are presented for preparing thin films of bR in a CaF2 cell and maintaining the hydration level between 0.1 and 0.5 μL/cm2, at a pH near 7.1. The same sample and same laser pulse energy were used to accumulate both IR and visible kinetic spectra. Visible spectra obtained both before and after co-addition of a series of IR spectra established that there was no deterioration of kinetics during exposure of the sample to many thousands of laser flashes required in the step scan data acquisition. The linear algebra-based procedures of Hendler et al. (2001) J. Phys. Chem. B, 105, 3319-3228 for obtaining clean absolute visible spectra of bacteriorhodopsin photocycle intermediates was adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra of these intermediates. Characteristic visible and IR markers were used to verify that the same kinetics were observed in both sets of data. Difference spectra made by subtracting sequential absolute spectra are shown for each transition based both on the homogeneous single cycle model and on the heterogeneous parallel cycles model. The latter has the two forms of M intermediate in separate linear pathways. All difference spectra are shown with peaks and troughs identified, as well as where the same IR features for an intermediate are seen both when it is a product and a precursor. A partial interpretation of these data is presented, but a much fuller analysis, in terms of the sequence of bindings and release of the proton during its transport across the membrane, changes in protein conformation or environment accompanying each transition, and identification of the most likely proton release group, is presented separately in the accompanying paper.
, Hendler, R.
, Smith, P.
and Kakareka, J.
Infrared and Visual Absolute and Difference Spectra of Bacteriorhodopsin Photocycle Intermediates Part 1: Isolation of Spectra, Applied Spectroscopy, [online], https://doi.org/10.1366/11-06302
(Accessed November 28, 2023)