Low-Temperature FTIR Study of Multiple K Intermediates in the Photocycles of Bacteriorhodopsin and Xanthorhodopsin

Abstract

Low-temperature FTIR spectroscopy of bacteriorhodopsin and xanthorhodopsin was used to elucidate the number of K-like bathochromic states, their sequence, and their contributions to the photo-equilibrium mixtures created by illumination at 80-180K. We conclude that in bacteriorhodopsin the photocycle includes three distinct K-like states in the sequence: bR→hvI∗→J→K0→KE→KL→L→…, and similarly in xanthorhodopsin. K0 is the main fraction in the mixture at 77K that is formed from J. K0 becomes thermally unstable above ~50K in both proteins. At 77K both J-to-K0 and K0-to-KE transitions occur and, contrarily to long-standing belief, cryogenic trapping at 77K does not produce a pure K state but a mixture of the two states, K0 and KE, with contributions from KE of ~15% and ~10% in the two retinal-proteins, respectively. Raising the temperature leads to increasing conversion of K0 to KE, and the two states coexist (without contamination from non K-like states) in the 80-140K range in bacteriorhodopsin, and in the 80-190K range in xanthorhodopsin. Temperature perturbation experiments in these regions of coexistence revealed that in spite of the observation of apparently stable mixtures of K0 and KE, the two states are not in thermally-controlled equilibrium. The K0-to-KE transition is unidirectional, and the partial transformation to KE is due to distributed kinetics, which governs the photocycle dynamics at temperatures below ~245K. From spectral deconvolution we conclude that the KE state, which is increasingly present at higher temperatures, is the same intermediate that is detected by time-resolved FTIR prior to its decay, on a time-scale of hundreds nanoseconds at ambient temperature, into the KL state. We were unable to trap the latter separately from KE at low temperature, due to the slow distributed kinetics and the increasingly faster overlapping formation of L state. Formation of the two consecutive K-like states in both proteins is accompanied by distortion of two different weakly bound water molecules: one in K0, the other in KE. The first, well-documented in bacteriorhodopsin at 77K where K0 dominates, was assigned to water 401 in bacteriorhodopsin. The other water molecule, whose participation has not been described previously, is disturbed on the next step of the photocycle, in KE, in both proteins. In bacteriorhodopsin the most likely candidate is water 407. However, unlike bacteriorhodopsin, the crystal structure of xanthorhodopsin lacks homologous weakly-bound water molecules.

Tuning the Primary Reaction of Channelrhodopsin-2 by Imidazole, pH, and Site-Specific Mutations

“…The equilibration between different K-states is supposed to be accompanied by a relaxation of the twisted chromophore leading to conformational changes in the protein, especially in the microenvironment of the retinal moiety. For bR, the transition between several K-like intermediates is also accompanied by the dislocation of weakly bound water molecules (51). These observations lead to the assumption that similar intermediates could also be present in the photocycle of ChR2.…”

Photocycle of Exiguobacterium sibiricum Rhodopsin Characterized by Low-Temperature Trapping in the IR and Time-Resolved Studies in the Visible

“…CLUSTAL W 62 , OPUS (Bruker, Germany), Matlab (The Mathworks, Inc., Natick, MA), KaleidaGraph (Synergy Software, Reading, PA), CurvExpert (http://www.curveexpert.net), FitExp 56 , and SchemeFit 56 software was used for data analysis and presentation.…”

“…Primary structure of ESR aligned in CLUSTAL W 62 with default settings. The 24 residues highlighted in yellow and cyan form the retinal-binding pocket in BR 52 ; out of those 18 (in yellow) are conserved, 6 (in cyan) are not.…”

Tracking Pore Hydration in Channelrhodopsin by Site-Directed Infrared-Active Azido Probes

“…Chromophore distortion is reflected by hydrogen out-of-plane (HOOP-) vibrations arising < 1000 cm -1 in the FTIR spectrum. Strong HOOP bands are observed in the K-like intermediate of bovine rhodopsin 146 , as well as the microbial rhodopsins BR and Xanthorhodopsin (XR) 147 , as well as PR 148 , Anabaena sensory rhodopsin 111 , phoborhodopsin 149 , or Exiguobacterium sibiricum rhodopsin 150 . In CrChR2 and GtCCR4, the HOOP bands are comparably weak 31,145 , while more pronounced in C1C2 and CaChR1 42,145,151 , raising the question whether the concept of energy storage is applicable to ChRs as well.…”

“…In the retinal fingerprint region, two negative bands at 1205/04 and 1166/65 cm -1 , both largely insensitive to H-D exchange, are present in both the red and the UV light induced spectra and hint at the depletion of a 13-trans,15-anti chromophore. 110 The band at 1166/65(-) cm -1 presumably reflects the υ(C10-C11) vibration of 13-trans retinal 144 and is not observed in CrChR2 31,103,151 and C1C2 42 but present in the greenabsorbing CaChR1 158 and ReaChR 51 , as well as BR 147 , implying differences in the chromophore geometry around the C10-C11 bond between Chrimson and CrChR2 that could be relevant for the bathochromic absorption shift. At both illumination conditions but more emphasized after UV illumination, two additional H-D sensitive negative bands are present at 1252 and 1234/33 cm -1 .…”