The N intermediate of bacteriorhodopsin at low temperatures: Stabilization and photoconversion

Balashov, Sergei P.; Imasheva, Eleonora S.; Litvin, F. F.; Lozier, Richard H.

doi:10.1016/0014-5793(90)80380-2

Cited by 18 publications

(28 citation statements)

References 9 publications

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“…Here we report on multiple distinct K intermediates. Unlike the three K states in 19 (see also 34 ) that differed in their convertibility by visible light of different wavelengths, the two K-like states here (Figure 6) exhibit little if any shift in the ethylenic band, and therefore are expected 82 to have the same visible absorption maximum.…”

Section: Discussionmentioning

confidence: 76%

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

2010

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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.

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Section: Discussionmentioning

confidence: 76%

“…The only evidence for more than two distinctly different K states was in 19 , where the interpretation focused on conformers formed in parallel from an inhomogeneous population of bacteriorhodopsin at cryogenic temperatures. Here we report on multiple distinct K intermediates.…”

Section: Discussionmentioning

confidence: 99%

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

2010

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“…Q (KN) is photochemically formed from the accumulated N [10][11][12][13]. The alkaline purple membrane suspension in the photo-stationary state is composed of bR568, M, N, and Q (and a small amount of O).…”

Section: Fluorescent Q and Non-fluorescent Nmentioning

confidence: 99%

“…The former efficiently fluoresced both in *Corresponding author. neutral and in acidic suspensions, and the latter, which is photochemically formed from N [10][11][12][13], fluoresced in alkaline and/ or high salt concentration suspension.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence spectra of bacteriorhodopsin and the intermediates O and Q at room temperature

et al. 1995

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“…Evidence that the assumption of a single state does not hold accumulated as K-like states were characterized by additional instrumental methods. Time-resolved and low temperature measurements in the visible 17–20 , UV 21,22 , IR 23–29 , resonance Raman 30–32 , CARS 33 , and photoelectric measurements 34 revealed distinct differences between several K-like states created under different conditions and characterized by different methods. From these differences the concept of a second K-like intermediate emerged.…”

Section: Introductionmentioning

confidence: 99%

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

Dioumaev

Lanyi

2009

J. Phys. Chem. B

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Time-resolved measurements were performed on wild-type bacteriorhodopsin with an optical multi-channel analyzer in the spectral range of 350–735 nm, from 100 ns to the photocycle completion, at four temperatures in the 5–30 °C range. The intent was to examine the possibility of two K-like bathochromic intermediates and to obtain their spectra and kinetics in the visible. The existence of a second K-like intermediate, termed KL, had been postulated (Shichida et al., BBA 723:240–246, 1983) to reconcile inconsistencies in data in the pico- and microsecond time-domains. However, introduction of KL led to a controversy since neither its visible spectrum nor its kinetics could be confirmed. Infra-red data (Dioumaev & Braiman, J. Phys. Chem., B, 101:1655–1662, 1997), revealed a state, which might have been considered a homologue to KL but it had a kinetic pattern different from that of the earlier proposed KL. Here we characterize two distinct K-like intermediates, KE (“early”) and KL (“late”), by their spectra and kinetics in the visible as revealed by global kinetic analysis. The KE-to-KL transition has a time constant of ~250 ns at 20 °C, and describes a shift from KE with λmax at ~600 nm and extinction of ~56,000 M−1·cm−1 to KL with λmax at ~590 nm and extinction of ~50,000 M−1·cm−1. The temperature dependence of this transition is characterized by an enthalpy of activation ΔH# ~ 40 kJ/mol, and a positive entropy of activation ΔS#/R = ~4. The consequences of multiple K-like states for interpreting the spectral evolution in the early stages of the photocycle are discussed.

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The N intermediate of bacteriorhodopsin at low temperatures: Stabilization and photoconversion

Abstract: In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl)

Cited by 18 publications

References 9 publications

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

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

Fluorescence spectra of bacteriorhodopsin and the intermediates O and Q at room temperature

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

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