Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

Dioumaev, Andrei K.; Lanyi, Janos Κ.

doi:10.1073/pnas.0703859104

Cited by 28 publications

(66 citation statements)

References 46 publications

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“…3d shows no evidence of the K state, although the sample has been cooled to 90 K for data acquisition. This is consistent with recent FTIR studies of cryogenic samples (24). Previous observations of K on cooling L (25) may have been due to irradiation of residual light-adapted bR, whereas in our experiments cooling and data acquisition occur in the dark.…”

Section: Resultssupporting

confidence: 81%

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.

193

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: Resultssupporting

confidence: 81%

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.

193

234

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“…5A). Thus, we conclude that the L 166 , L 174 , and L 181 states contribute to the L-to-bR back-reaction first detected by lowtemperature optical studies (22,23) and recently confirmed by IR spectroscopy (24). However, because the persistent L 186 relaxes to M at still-higher temperatures, we can confidently conclude that this is the functional L (19).…”

Section: Discussionmentioning

confidence: 89%

Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization–enhanced solid-state NMR

Bajaj

Mak‐Jurkauskas²,

Belenky³

et al. 2009

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

302

383

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Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of Ϸ90. Multidimensional spectroscopy of U-13 C, 15 N-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR568 and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.ultidimensional magic-angle spinning (MAS) solid-state NMR is a general tool in structural studies of membrane proteins that are inaccessible to crystallography and solutionstate NMR, as demonstrated by recent successful applications (1-3). But the sensitivity of these experiments is low, which becomes a significant problem when multidimensional experiments are needed to characterize systems of higher molecular weight. The sensitivity deficit is even more acute when NMR signals are further divided among multiple states, as is often the case for trapped reaction intermediates. Consequently, a 1-2 order of magnitude enhancement of NMR sensitivity is essential for applications of multidimensional MAS NMR methods to studies of reaction intermediates of membrane proteins.One approach to improving the sensitivity of NMR is dynamic nuclear polarization (DNP), in which the Ϸ660-fold greater spin polarization of unpaired electrons in a paramagnetically doped glassy matrix is transferred to nuclei before an NMR experiment (4). Here, we demonstrate that high-frequency DNP by using a stable, high-power 250 GHz microwave source (5) and an efficient, nonperturbing biradical polarizing agent (6, 7), is a potentially general approach for biological MAS NMR. A 43-fold signal enhancement from DNP, combined with operation at 90 K, yields an overall 90-fold signal enhancement over previous experiments at 183 K (8). The resulting Ϸ8,100-fold savings in acquisition time permits 2-dimensional (2D) resolution of signals from mixtures of reaction intermediates that would be impossible to observe absent the enhancement available from DNP.In bacteriorhodopsin (bR), 7 transmembrane helices surround a transport channel in whic...

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“…trapped at cryogenic temperature) 15,35,41-44,46-48 and time-resolved 23-30,36-40,45 techniques. Detailed assignment of individual vibrational modes was through extensive use of isotope-labeled retinal incorporated into bacteriorhodopsin by Raman 49 (and most fully summarized in 50 ) and by FTIR 44,46,51 methods, creating the basis for studies of other retinal-proteins.…”

Section: Introductionmentioning

confidence: 99%

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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Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

Cited by 28 publications

References 46 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

Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization–enhanced solid-state NMR

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

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