Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy

Diller, Rolf; Iannone, Mark A.; Cowen, B. R.; Maiti, Sudipta; Bogomolni, Roberto A.; Hochstrasser, Robin M.

doi:10.1021/bi00139a020

Cited by 50 publications

(40 citation statements)

References 34 publications

(68 reference statements)

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“…19 A time-resolved infrared spectrum of the K intermediate recorded 10 ns after photolysis at room temperature shows no bands in the spectral region of the carboxyl groups. 24 This spectrum, which was obtained by use of a tunable CO laser, had a detection limit better than 1 × 10 -4 OD units. It was concluded that a conformational change of the carboxylic residues occurs after 10 ns but before 500 ns.…”

Section: Discussionmentioning

confidence: 92%

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

et al. 1996

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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.

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Section: Discussionmentioning

confidence: 92%

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

et al. 1996

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“…12,13 On the other hand, picosecond time-resolved resonance Raman spectroscopy revealed that the K-KL transition occurs with a time constant of ∼70 ps, 15 whereas timeresolved infrared spectroscopy reported that the conversion time is up to 50 ps. 21 The subsequent intermediate, L, is formed in ∼2 µs. 13 All the tryptophan bands probed at 225 nm ( Figure 2) as well as the Y8a band in the 238 nm spectra ( Figure 4) bleached with in the instrumental response time.…”

Section: Discussionmentioning

confidence: 99%

Picosecond Time-Resolved Ultraviolet Resonance Raman Spectroscopy of Bacteriorhodopsin: Primary Protein Response to the Photoisomerization of Retinal

et al. 2009

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Protein dynamics in the primary processes during the bacteriorhodopsin (BR) photocycle under physiological conditions were investigated by measuring picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of the BR suspended solution at ambient temperature. We used a 565 nm pump pulse to initiate the BR photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, that is, Trp86, Trp182, Tyr57, and Tyr185. The 225 nm UVRR spectra exhibited bleaching of intensity for all the tryptophan bands within the instrumental response, followed by recovery with a time constant of 30 ps and no further changes up to 1 ns. This suggests that the stepwise structural changes in the tryptophan residues proceed in response to the retinal photoreaction. It is concluded that the initial intensity bleach arises from the J-intermediate formation and the 30 ps recovery is associated with the K-KL transition. The 30 ps process in the BR photocycle has been detected for the first time. In the 238 nm UVRR spectra, spectral features attributable to the K and KL intermediates were observed. The observed spectral changes showed that the temporal behaviors of the observed spectral changes in each Raman band of both tryptophan and tyrosine were different. This indicates that the spectral changes originated from structural changes of at least two tryptophan and two tyrosine residues.

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“…Its existence is required to reconcile the time-resolved 29 and the low-temperature FTIR data 23,79 . A time constant of 40–100 ps was proposed for such a transition based on fast measurements in Raman 32 , IR 25 and UV 22 . Such 70±30 ps 22,25 time constant is consistent with conclusions from the photodamage experiments 45 .…”

Section: Discussionmentioning

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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Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy

Cited by 50 publications

References 34 publications

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

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

Picosecond Time-Resolved Ultraviolet Resonance Raman Spectroscopy of Bacteriorhodopsin: Primary Protein Response to the Photoisomerization of Retinal

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

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