Time-Resolved Step-Scan FT-IR Investigations of the Transition from KL to L in the Bacteriorhodopsin Photocycle: Identification of Chromophore Twists by Assigning Hydrogen-Out-Of-Plane (HOOP) Bending Vibrations

Weidlich, O.; Siebert, Friedrich

doi:10.1366/0003702934067351

Cited by 108 publications

(139 citation statements)

References 44 publications

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“…However, C 15 -HOOP signals in K (1-4) and L (6)(7)(8) suggest that the locus of the distortion is at C 15 in both intermediates and, therefore, that the singlebond torsion in K is primarily around the C 14 -C 15 bond and the double-bond torsion in L is primarily around the C 15 ϭN bond. In any case, the results are difficult to reconcile with crystal structures.…”

Section: Resultsmentioning

confidence: 99%

Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR

Mak‐Jurkauskas

Bajaj²,

Hornstein

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

198

234

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Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult, as judged by the multiple L substates, with weaker counterion interactions, that are trapped at lower temperatures. The doublebond torsion ultimately developed in the first half of the photocycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer.energy transduction ͉ photocycle intermediates ͉ dynamic nuclear polarization ͉ ion transport ͉ retinal protein T he light-driven ion pump, bacteriorhodopsin (bR), has been studied extensively since it was discovered in the 1970s. Its availability and stability have made it the prototypical transmembrane protein, ion pump, retinal pigment, and model for G protein-coupled receptors. As such, it has been the target of a wide variety of biophysical techniques that have garnered a great deal of information about the structure of the protein and the changes that it undergoes during its functional photocycle. Nevertheless, it remains unclear how the protein stores and channels energy to translocate ions and prevent backflow.An important feature of the pump cycle ( Fig. 1) is that the change in connectivity of the active site between the two sides of the membrane occurs midway through the photocycle (in the transition from the early M state to the late M state), long after the initial photoisomerization of the retinylidene chromophore from all-trans to 13-cis (Fig. 2), and long before the thermal reisomerization of the chromophore at the end of the photocycle. Because the change in connectivity is divorced from the major isomerization events, much attention has been directed to the process(es) that might be responsible. However, in the fuller context, the more interesting question is how the active site remains connected to the extracellular surface for so long after the photoisomerization event, and what finally releases it from that set of interactions. In this light, it is not surprising that vibrational spectroscopy finds indications of a strained chromophore in the K (1-4) and L (5-8) intermediates, and a relaxed chromophore in the N intermediate (9). Evidence of strain is also seen in magic angle spinning (MAS) NMR spectra. Furthermore, MAS NMR has pinpointed the release of this strain to the transition from early M to late M (i.e., coincident with the connectivity change) and determin...

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Section: Resultsmentioning

confidence: 99%

Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR

Mak‐Jurkauskas

Bajaj²,

Hornstein

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

198

234

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…[1][2][3][4] While the resonance Raman technique has dominated this research area thus far, new scientific issues are emerging in the field of chemical and biological reactions that are effectively addressed by transient infrared spectroscopy. One such new theme is the involvement of the surrounding matrix in controlling chemical and biological reaction pathways.…”

Section: Introductionmentioning

confidence: 99%

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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“…The control of the photo-induced reaction can be done (i) kinetically, by recording the spectrum during a short time after a lightflash or by using time-resolved FTIR difference spectroscopy; (ii) by controlling the sample temperature; (iii) by using chemicals (electron donors and/or electron acceptors, reducing or oxidizing agents) to poise a light-induced reaction intermediate. Time-resolved FTIR spectroscopy involves rapid scan techniques, with 10-25 ms time resolution or step-scan FTIR spectroscopy (Weidlich and Siebert 1993) with time resolution in the ls to ns range (Mezzetti et al 2003;Kötting and Gerwert 2005;Sivakumar et al 2005). In addition, timeresolved IR spectroscopy can be achieved using tunable infrared laser diodes .…”

Section: Light-induced Ftir Difference Spectroscopymentioning

confidence: 99%

Fourier transform infrared (FTIR) spectroscopy

2009

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Fourier transform infrared (FTIR) spectroscopy probes the vibrational properties of amino acids and cofactors, which are sensitive to minute structural changes. The lack of specificity of this technique, on the one hand, permits us to probe directly the vibrational properties of almost all the cofactors, amino acid side chains, and of water molecules. On the other hand, we can use reaction-induced FTIR difference spectroscopy to select vibrations corresponding to single chemical groups involved in a specific reaction. Various strategies are used to identify the IR signatures of each residue of interest in the resulting reaction-induced FTIR difference spectra. (Specific) Isotope labeling, site-directed mutagenesis, hydrogen/deuterium exchange are often used to identify the chemical groups. Studies on model compounds and the increasing use of theoretical chemistry for normal modes calculations allow us to interpret the IR frequencies in terms of specific structural characteristics of the chemical group or molecule of interest. This review presents basics of FTIR spectroscopy technique and provides specific important structural and functional information obtained from the analysis of the data from the photosystems, using this method.

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Time-Resolved Step-Scan FT-IR Investigations of the Transition from KL to L in the Bacteriorhodopsin Photocycle: Identification of Chromophore Twists by Assigning Hydrogen-Out-Of-Plane (HOOP) Bending Vibrations

Cited by 108 publications

References 44 publications

Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR

Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR

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

Fourier transform infrared (FTIR) spectroscopy

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