Chromophore Distortions in the Bacteriorhodopsin Photocycle: Evolution of the H−C14−C15−H Dihedral Angle Measured by Solid-State NMR † (original) (raw)
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Biochemistry, 1997
The L 550 intermediate in the bacteriorhodopsin (bR) photocycle has drawn much attention with respect to the mechanism of light-driven proton transport because it selectively releases the Schiff base (SB) proton to the extracellular proton channel in the LfM transition. Here we extend our solidstate NMR studies of bR photocycle intermediates to the L state. Under conditions that stabilize L 550 , a new SB signal is detected in the 15 N NMR spectrum which disappears upon thermal relaxation. This signal is in the range for a protonated SB, but downfield from the SB signals of bR 568 and N 520. Since steric interactions would have the opposite effect on shielding, the data argue against a 13,14-dicis chromophore in L 550. Comparison with the 15 N chemical shifts of halide salts of protonated Schiff bases (pSB's) of retinal suggests that the interaction of the SB with its counterion is significantly stronger in L 550 than in N 520 (which in turn is stronger than in bR 568). This is consistent with models of the early photocycle in which the electrostatic interaction between the SB and its counterion constitutes an important constraint. Although the L counterion interaction is comparable to that of a 6-s-trans,13-cis chloride salt, the visible spectrum is strongly red-shifted from the λ max) 491 nm of the chloride. This suggests some double bond strain in L 550 , particularly about the CdN bond. This strain is apparently gone in the N intermediate, which has a normal relationship between the 15 N chemical shift and λ max. Such a relaxed chromophore is consistent with orientation of the SB proton toward the cytoplasmic surface in the N intermediate.
Proceedings of the National Academy of Sciences of the United States of America, 2008
By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The (15)N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. 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 n...
Proceedings of The National Academy of Sciences, 2000
We measured the M-N transition of wild-type bacteriorhodopsin (pH 9, 10°C) by time-resolved x-ray diffraction study at SPring8 BL45XU-A. We confirmed the accumulation of M and N intermediates by absorbance measurements, and we found that the time resolution of x-ray diffraction experiments (244 ms) was sufficient to resolve the M-N transition. From the x-ray diffraction data, three components were decomposed by singular value decomposition analysis. The existence of three components in the M3 N3 BR reaction revealed that BR changes its structure during the M-N transition. Moreover, the difference Fourier maps of reconstituted fast and slow decay components clearly showed that the electron density distributions of the F helix changes in the M-N transition. The observed structural change at the F helix will increase access of the Schiff base and D96 to the cytoplasmic surface and facilitate the proton transfer steps that begin with the decay of the M state.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000
Light-induced isomerization leads to orientational changes of the retinylidene chromophore of bacteriorhodopsin in its binding pocket. The chromophore reorientation has been characterized by the following methods: polarized absorption spectroscopy in the visible, UV and IR; polarized resonance Raman scattering ; solid-state deuterium nuclear magnetic resonance; neutron and X-ray diffraction. Most of these experiments were performed at low temperatures with bacteriorhodopsin trapped in one or a mixture of intermediates. Time-resolved measurements at room temperature with bacteriorhodopsin in aqueous suspension can currently only be carried out with transient polarized absorption spectroscopy in the visible. The results obtained to date for the initial state and the K, L and M intermediates are presented and discussed. The most extensive data are available for the M intermediate, which plays an essential role in the function of bacteriorhodopsin. For this intermediate the various methods lead to a consistent picture : the curved all-trans polyene chain in the initial state straightens out in the M intermediate (13-cis) and the chain segment between C 5 and C 13 tilts upwards in the direction of the cytoplasmic surface. The kink at C 13 allows the positions of L-ionone ring and Schiff base nitrogen to remain approximately fixed. ß
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000
In recent years, significant progress has been made in elucidating the structure of bacteriorhodopsin. However, the molecular mechanism by which vectorial proton motion is enforced remains unknown. Given the advantages of a protonated Schiff base for both photoisomerization and thermal reisomerization of the chromophore, a five-state proton pump can be rationalized in which the switch in the connectivity of the Schiff base between the two sides of the membrane is decoupled from double bond isomerization. This decoupling requires tight control of the Schiff base until it is deprotonated and decisive release after it is deprotonated. NMR evidence has been obtained for both the tight control and the decisive release: strain develops in the chromophore in the first half of the photocycle and disappears after deprotonation. The strain is associated with a strong interaction between the Schiff base and its counterion, an interaction that is broken when the Schiff base deprotonates. Thus the counterion appears to play a critical role in energy transduction, controlling the Schiff base in the first half of the photocycle by`electrostatic steering'. NMR also detects other events during the photocycle, but it is argued that these are secondary to the central mechanism. ß
Evidence for Charge-Controlled Conformational Changes in the Photocycle of Bacteriorhodopsin
Biophysical Journal, 1998
The existence of two different M-state structures in the photocycle of the bacteriorhodopsin mutant ASP38ARG was proved. At pH 6.7 (0 to −6°C) a spectroscopic M intermediate (M1) that does not differ significantly in its tertiary structure from the light-adapted ground state accumulates under illumination. At pH > 9 another state (M2), characterized by additional pronounced changes in the Fourier transform infrared difference spectrum in the region of the amide I and II bands, accumulates. The M2 intermediate trapped at pH 9.6 displays the same changes in the x-ray diffraction intensities under continuous illumination as previously described for x-ray experiments with the mutant ASP96ASN. These observations indicate that in this mutant the altered charge distribution at neutral pH controls the tertiary structural changes that seem to be necessary for proton translocation.
Protein conformational changes in the bacteriorhodopsin photocycle
Journal of Molecular Biology, 1999
We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Speci®c intermediates that accumulate in the late stages of the photocycle of wildtype bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light¯ash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A Ê resolution to de®ne light-driven changes in protein conformation.
Biophysical Journal, 1999
According to the current structural model of bacteriorhodopsin, Ile 222 is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant Ile 222 3 Cys with p-chloromercuribenzoic acid and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the M N photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the difference Fourier maps between the M N intermediate and the unphotolyzed state, the structural change in the M N intermediate was not affected by mercury labeling. The difference Fourier map between the labeled and the unlabeled I222C gave the position of the mercury label. This information was obtained for both the unphotolyzed state and the M N intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1 Ϯ 0.8 Å in the M N intermediate. This agrees with earlier results that suggested a structural change in the G helix. The movement of the mercury label is so large that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 222. Because Ile 222 is located at the same level on the z coordinate as Asp 96 , the structural change in the G helix could have the functional role of perturbing the environment and therefore the pK a of this functionally important aspartate.
Structural Changes during the Formation of Early Intermediates in the Bacteriorhodopsin Photocycle
Biophysical Journal, 2002
Early intermediates of bacteriorhodopsin's photocycle were modeled by means of ab initio quantum mechanical/molecular mechanical and molecular dynamics simulations. The photoisomerization of the retinal chromophore and the formation of photoproducts corresponding to the early intermediates were simulated by molecular dynamics simulations. By means of the quantum mechanical/molecular mechanical method, the resulting structures were refined and the respective excitation energies were calculated. Two sequential intermediates were found with absorption maxima that exhibit red shifts from the resting state. The intermediates were therefore assigned to the K and KL states. In K, the conformation of the retinal chromophore is strongly deformed, and the NOH bond of the Schiff base points almost perpendicular to the membrane normal toward Asp-212. The strongly deformed conformation of the chromophore and weakened interaction of the Schiff base with the surrounding polar groups are the means by which the absorbed energy is stored. During the K-to-KL transition, the chromophore undergoes further conformational changes that result in the formation of a hydrogen bond between the NOH group of the Schiff base and Thr-89 as well as other rearrangements of the hydrogen-bond network in the vicinity of the Schiff base, which are suggested to play a key role in the proton transfer process in the later phase of the photocycle.