Nanosecond time-resolved infrared spectroscopy distinguishes two K species in the bacteriorhodopsin photocycle

Abstract

The photochemical reaction process of bacteriorhodopsin in the nanosecond time range (-120-860 ns) was measured in the 1400-900 cm-1 region with an improved time resolved dispersive-type infrared spectrometer. The system is equipped with a newly developed detection unit whose instrumental response to a 5-ns laser pulse has a full width of the half-maximum of 60 ns. It provides highly accurate data that enabled us to extract a kinetic process one order of magnitude faster than the instrumental response. The spectral changes in the 1400-900 cm-1 region were analyzed by singular value decomposition and resolved into three components. These components were separated by fitting with 10- and 1000-ns exponential functions and a step function, which were convoluted with the instrumental response function. The components with decay time constants of 10 and 1000 ns are named K and KL, respectively, on the basis of previous visible spectroscopy. The spectral shapes of K and KL are distinguishable by their hydrogen-out-of-plane (HOOP) modes, at 958 and 984 cm-1, respectively. The former corresponds to the K intermediate recorded at 77 K and the latter to a K-like photoproduct at 135 K. On the basis of published data, these bands are assigned to the 15-HOOP mode, indicating that the K and KL differ in a twist around the C14-C15 bond.

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

“…These studies were carried out on hydrated films of BR. 12,13 It is therefore possible that the fast structural change, occurring with a time constant of 400 ns, was not observed in the previous studies because it is somehow blocked in the hydrated films. Another explanation would be that it could not be detected because of insufficient spectral quality and time resolution.…”

“…It was observed that this conversion is reversible 2,12,13,18 and that the ratio of the rate constants k LfKL /k KLfL increases with dehydration. 18 To examine the spectral changes with time, our 0-200, 200-400, 400-600, and 600-800 ns FTIR difference spectra are shown directly overlayed in Figure 2.…”

“…The band at 1740 cm -1 in this 0-20 ns spectrum has an intensity of about 5 × 10 -4 OD units. On the basis of time constants of about 10 ns and 1 µs for the KfKL and KLfL transitions, 12 respectively, the 0-20 ns spectrum corresponds to a mixture of K and KL and the 0-100 ns spectrum to the KL intermediate. The fact that the band structure at 1740 cm -1 in these two spectra is nearly identical to the low-temperature BR/K spectrum implies that the local environment of Asp-115 in the K and KL intermediates in the BR suspension at room temperature are similar to K at low temperature and that a perturbation of this residue already occurs during the formation of K. This also implies a direct coupling of Asp-115 to the chromophore, which is consistent with the result that the dynamics of Asp-96 and Asp-115 as well as of the faster responding modes of the chromophore show the same time constant.…”

“…10,11 A further transition to KL exhibits a time constant of about 10 ns, 12 while the L intermediate is formed in about 2 µs. [12][13][14][15] The KfKL and KLfL conversions are steps in which energy stored in the distorted chromophore is transferred to the protein. 12 The elucidation of the conformational changes of the chromophore as well as of the protein in the KL to L transition is crucial for developing an understanding of the photocycle, because L is a key intermediate leading to proton transfer in the L to M conversion.…”

Water Structural Changes in the L and M Photocycle Intermediates of Bacteriorhodopsin as Revealed by Time-Resolved Step-Scan Fourier Transform Infrared (FTIR) Spectroscopy

“…Figure 5 shows an L-minus-BR spectrum (a) that was obtained by subtracting the M-minus-BR spectrum (20% of b 3 ) and the contribution of K L (50% of b 0 ) from b 1 as described above, together with the L-minus-BR spectrum at 170 K (b). The spectrum at 170 K was obtained as described previously (27).…”