Water molecules and exchangeable hydrogen ions at the active centre of bacteriorhodopsin localized by neutron diffraction

Παπαδόπουλος, Γεώργιος; Dencher, Norbert A.; Zaccaı̈, Giuseppe; ̈ldt, Georg Bu

doi:10.1016/0022-2836(90)90140-h

Cited by 171 publications

(119 citation statements)

References 25 publications

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“…Internal water, protein side groups, and the backbone might contribute hydrogenbonds to such a network. There is experimental evidence for internal bound water in bR (34)(35)(36)(37). Molecular dynamics calculations on the bR structural model proposed by Henderson et at (13) orient a chain of water molecules just between D96 and the Schiff base as presented in the scheme (38,39).…”

Section: Methodsmentioning

confidence: 99%

Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Coutre

Tittor

Oesterhelt

et al. 1995

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

107

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Experimental evidence for proton transfer via a hydrogen-bonded network in a membrane protein is presented. Bacteriorhodopsin's proton transfer mechanism on the proton uptake pathway between Asp-96 and the Schiff base in the M-to-N transition was determined. The slowdown of this transfer by removal of the proton donor in the Asn mutant can be accelerated again by addition of small weak acid anions such as azide. Fourier-transform infrared experiments show in the Asp-96 -k Asn mutant a transient protonation of azide bound to the protein in the M-to-N transition and, due to the addition of azide, restoration of the IR continuum band changes as seen in wild-type bR during proton pumping. The continuum band changes indicate fast proton transfer on the uptake pathway in a hydrogen-bonded network for wild-type bR and the Asp-96 Asn mutant with azide. Since azide is able to catalyze proton transfer steps also in several kinetically defective bR mutants and in other membrane proteins, our finding might point to a general element of proton transfer mechanisms in proteins.The retinal protein bacteriorhodopsin (bR), probably the best understood example of an ion pump, is a suitable system to study intramolecular proton transfer in proteins (1). Crucial events for the transport mechanism during the photocycle via the intermediates J, K, L, M, N, and 0 are the light-induced isomerization of all-trans-retinal to 13-cis-retinal in the BRto-J photoreaction (where BR is the ground state of the bR molecule), the deprotonation of the Schiff base which is the binding site between chromophore and protein, (2-4), and protonation changes of internal aspartic acids (5). Fouriertransform infrared (FTIR) spectroscopy of mutant bRs identified the proton acceptor on the proton release pathway as Asp-85 (D85) (6-8) and the proton donor on the proton uptake pathway as D96 (8). The proton acceptor D85 is protonated in the L-to-M reaction, in which the Schiff base becomes deprotonated, whereas the donor D96 is deprotonated in the M-to-N transition, in which the Schiff base is reprotonated (9). Evidence for the catalytic role of these aspartic acids is contributed.by electrical charge displacement measurements showing disturbed proton transport in D85X and D96X mutants (10, 11). These results on the reaction mechanism were strongly confirmed in all details in a series of succeeding publications (for review, see ref. 12). The atomic structural model of bR as developed from high-resolution electron microscopic studies proposed the positions of D85 and D96 on either side of the retinal (13). Therefore they are correctly located for their role in the vectorial proton translocation process.However, the donor, D96, is positioned in the structural model about 12 A from the acceptor, the Schiff base (13). Even though the resolution of the structure in the cross section of the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S...

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Section: Methodsmentioning

confidence: 99%

Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Coutre

Tittor

Oesterhelt

et al. 1995

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

107

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“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.The structure and hydration of PM have been studied as a function of temperature and relative humidity by neutron diffraction (11,12). Progressive drying of the membranes showed that the hydration around the lipid headgroups was removed first.…”

mentioning

confidence: 99%

Thermal motions and function of bacteriorhodopsin in purple membranes: effects of temperature and hydration studied by neutron scattering.

Ferrand

Dianoux²,

Petry³

et al. 1993

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

398

362

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The internal dynamics of bacteriorhodopsin, the light-driven proton pump in the purple membrane of Halobacterium halobium, has been studied by inelastic neutron scattering for various conditions oftemperature and hydration. Light activation can take place when the membrane is vibrating harmonically. The ability of the protein to functionally relax and complete the photocycle initiated by the absorption of a photon, however, is strongly correlated with the onset of low-frequency, large-amplitude anharmonic atomic motions in the membrane. For a normally hydrated sample, this occurs at about 230 K, where a dynamical transition from a lowtemperature harmonic regime is observed. In moderately dry samples, on the other hand, in which the photocycle is slowed down by several orders of magnitude, no transition is observed and protein motions remain approximately harmonic up to room temperature. These results support the hypothesis, made from previous neutron diffraction studies, that the "softness" of the membrane modulates the function of bacteriorhodopsin by allowing or not allowing large-amplitude motions in the protein.The direct environment of a protein has a strong influence on protein internal dynamics and function. Inelastic neutron scattering has unique advantages for the study of thermal motions in proteins. Here we describe such a study of a membrane protein in its natural lipid environment under various external conditions. By varying temperature and hydration, the thermal motions of bacteriorhodopsin (BR) in the purple membrane (PM) of Halobacterium halobium were characterized and correlated with aspects ofprotein function. Previously, thermal dynamics had been studied mainly in small globular proteins by various experimental methods, such as Mossbauer spectroscopy (1), inelastic neutron scattering (2) or optical spectroscopy (3), used to sample their atomic motions, as well by molecular dynamics simulation approaches (4).PM functions as a light-driven proton pump. It contains a single protein of 26 kDa, BR, organized with lipid on a highly ordered two-dimensional lattice. The structure of BR is now known to relatively high resolution for a membrane protein in its natural lipid environment (5). Its main features are seven a-helices, arranged in a bundle around a retinal molecule bound via a Schiff base to a lysine residue in the protein.Upon illumination, PM undergoes a "photocycle" with a frequency of the order of milliseconds (6). The photocycle has been studied extensively and was shown to be greatly influenced by external conditions such as temperature and relative humidity (7-10).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.The structure and hydration of PM have been studied as a function of temperature and relative humidity by neutron diffraction (11,12). Progressive drying of the membranes showed that the hydration around the lipid...

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“…Zaccai has suggested that the inhibition of bR activity at low temperatures or in the dry state could be due to a reduction of motions from the close packing of lipids around the protein in PMs (47). Not only the interbilayer spacing but also the in-plain unit cell parameter (defined as the length of the trimer unit of bR in the lattice) decreases about 1 Å for the completely dried film compared with that of a fully hydrated film (15,47,48,77,78). The decay of the M-intermediate in the photocycle and the regeneration of the ground state are strongly slowed below 80% rh (46).…”

Section: Dynamics Of Phospholipids In the Pmmentioning

confidence: 99%

“…Saturated KCl solution was used to control the rh to 85% at 293 K (52) for five days. PM films formed on the thin glass plates or some other supports have been used for the structural and functional determination of bR by several spectroscopic and diffraction methods without recourse to further crystallization (47,48,53,54). The lipid bilayers stack uniaxially parallel to the plates together with intercallating water layers to preserve the directional quality of the protein and to maintain the structural and functional integrity under a wide range of conditions, such as pH, temperature, humidity, and chemical environment (55).…”

mentioning

confidence: 99%

Functionally Relevant Coupled Dynamic Profile of Bacteriorhodopsin and Lipids in Purple Membranes

Kamihira

Watts

2006

Biochemistry

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The dynamics of bacteriorhodopsin (bR) and the lipid headgroups in oriented purple membranes (PMs) was determined at various temperatures and relative humidity (rh) using solid-state NMR spectroscopy. The 31 P NMR spectra of the R-and γ-phosphate groups in methyl phosphatidylglycerophosphate (PGP-Me), which is the major phospholipid in the PM, changed sensitively with hydration levels. Between 253 and 233 K, the signals from a fully hydrated sample became broadened similarly to those of a dry sample at 293 K. The 15 N cross polarization (CP) NMR spectral intensities from [ 15 N]Gly bR incorporated into fully hydrated PMs were suppressed in 15 N CP NMR spectra at 293 K compared with those of dry membranes but gradually recovered at low temperatures or at lower hydration (75%) levels. The suppression of the NMR signals, which is due to interference with proton decoupling frequency (∼45 kHz), coupled with short spin-spin relaxation times (T 2 ) indicates that the loops of bR, in particular, have motional components around this frequency. The motion of the transmembrane R-helices in bR was largely affected by the freezing of excess water at low temperatures. While between 253 and 233 K, where a dynamic phase transition-like change was observed in the 31 P NMR spectra for the phosphate lipid headgroups, the molecular motion of the loops and the C-and N-termini slowed, suggesting lipidloop interactions, although protein-protein interactions between stacks cannot be excluded. The results of T 2 measurements of dry samples, which do not have proton pumping activity, were similar to those for fully hydrated samples below 213 K where the M-intermediates can be trapped. These results suggest that motions in the 10s µs correlation regime may be functionally important for the photocycle of bR, and protein-lipid interactions are motionally coupled in this dynamic regime.Bacteriorhodopsin (bR) 1 is one of the most extensively studied light-driven proton pumps in Haloarchaea. It contains seven transmembrane R-helices surrounding the retinal chromophore and has been the structural paradigm of G-protein-coupled receptors and other membrane proteins (1-5). It forms a protein-lipid complex of defined composition in the purple membrane (PM), where bR is arranged in trimeric units assembled in 2D crystalline patches in a hexagonal lattice. High-resolution X-ray (6-11) and electron (12, 13) crystallography have been used to determine the 3D structure of bR and the structural changes at K, L, M, N, and O photointermediates to understand the unidirectional translocation of proton pumping activity (14-23). Solid-state NMR spectroscopy has been used to provide detailed information about the local structural changes of retinal (24,25) around the key residues involved in proton pumping activity (26,27) and the local mobility of bR (28-30) under physiological conditions. Also, the structure and orientation of some R-helices of membrane-embedded bR have been determined by polarization inversion spin exchange at the magic angle (PISE...

show abstract

Water molecules and exchangeable hydrogen ions at the active centre of bacteriorhodopsin localized by neutron diffraction

Cited by 171 publications

References 25 publications

Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Thermal motions and function of bacteriorhodopsin in purple membranes: effects of temperature and hydration studied by neutron scattering.

Functionally Relevant Coupled Dynamic Profile of Bacteriorhodopsin and Lipids in Purple Membranes

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