Protein Dynamics in the Bacteriorhodopsin Photocycle:  A Nanosecond Step-Scan FTIR Investigation of the KL to L Transition

Abstract

A time-resolved step-scan FTIR spectrometer with a time resolution of 20 ns was developed and used to investigate the KL to L transition in the photocycle of bacteriorhodopsin in the time range from -60 to 940 ns. Broadband FTIR absorbance difference spectra with a spectral range of 850-2050 cm -1 and a spectral resolution of 4 cm -1 have been obtained. Our data show that there are two sets of BR photoproduct difference bands exhibiting different kinetics. The intensity changes of bands attributed to structural changes of the carboxyl groups Asp-96 and Asp-115 as well as of bands assigned to CdC and CsC stretching modes of the chromophore show single exponential behavior with a time constant of about 400 ns, implying that these two processes are coupled. The time-dependent intensity changes of bands attributed to structural changes of the chromophore region near the Schiff base exhibit slower kinetics with a time constant of about 2 µs. We interpret our data in terms of a process during the KL to L transition where structural changes of the -ionone ring end of the chromophore and of Asp-115 occur faster than changes at the Schiff base region of the chromophore. Under our physiological sample conditions, a perturbation of Asp-115 occurs in the first 20 ns in contrast to results from hydrated films where this process is blocked or occurs more slowly. This fast protein response indicates that there is direct coupling between the carboxylic acid residues and the chromophore. Comparison of our data with low-temperature and microsecond time-resolved infrared spectra of the L intermediate in hydrated films of BR indicates that a different L structure is produced when the water activity is low.

Time resolved infrared studies of C-H bond activation by organometallics

“…[lo] In a standard FTIR, light from a broadband 15 is the spectrum that we actually want to see, and it has features that are on the order of .5 mOD, or 0.05% of the static spectrum. If we assume an 8 bit digitizer, which has 2*=256, the difference between two digitized values is & = .00390, or .4%.…”

Two Bathointermediates of the Bacteriorhodopsin Photocycle, from Time-Resolved Nanosecond Spectra in the Visible

“…Thus, on the one hand, the data obtained by a variety of methods did suggest existence of an additional intermediate between the K state that is formed in picoseconds and L (i.e. a state ‘homologous’ to KL) 17–19,21–27,29–34 , but on the other hand, it was shown 35,36 that this state can have neither the spectrum nor kinetics, which would fit the KL state in 19 . Use of the nonexistent ~10 ns time constant to re-calculate spectra from the data-sets that did not allow an independent estimate of kinetics 26,27,37 created further confusion.…”

A Role for Internal Water Molecules in Proton Affinity Changes in the Schiff Base and Asp85 for One‐way Proton Transfer in Bacteriorhodopsin†

“…The visible spectrum of L 1 , which arises in the sub‐microsecond time domain before the appearance of normal L (presumably L 2 from the time constant), shows a more substantial tail toward longer wavelengths, like L at low temperature (30), probably due to some distortion around the Schiff base. The FTIR spectrum in a time range similar to that for L 1 shows changes in the bands due to the chromophore and Asp96/Asp115 similar to those in L, which occur in advance of the Schiff base changes observed in normal L (55). However, this precursor of L did not show changes in vibrational bands characteristic of those of L at low temperatures: the Schiff base bands at 1310 and 1075/1064/1056 cm −1 .…”