Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle

Balashov, Sergei P.; Govindjee, Rajni; Kono, Masahiro; Imasheva, Eleonora S.; Лукашев, Е. П.; Ebrey, Thomas G.; Crouch, Rosalie K.; Menick, Donald R.; Ying-jun, Feng

doi:10.1021/bi00090a008

Cited by 179 publications

(370 citation statements)

References 58 publications

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“…The effect of the arginine 82 to alanine mutation on dark adaptation, proton release, and photochemical cycle in membrane fragments was analyzed by Balashov et al (21). These authors confirmed the reversed order of proton release and uptake observed in Ref.…”

Section: Kinetics Of Proton Release and Uptakementioning

confidence: 57%

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Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

Alexiev¹,

Mollaaghababa²,

Khorana³

et al. 2000

Journal of Biological Chemistry

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Evidence is presented for long range interactions between the extracellular and cytoplasmic parts of the heptahelical membrane protein bacteriorhodopsin in the mutant R82A and its second site revertant R82A/ G231C. (i) In the double mutants R82A/G72C and R82A/ A160C, with the cysteine mutation on the extracellular or cytoplasmic surface, respectively, the photocycle is the same as in the single mutant R82A with an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake. Proton release and uptake kinetics were measured directly at either surface by using the unique cysteine residue as attachment site for the pH indicator fluorescein. Whereas in wild type proton uptake on the cytoplasmic surface occurs during the M-decay ( ϳ 8 ms), in R82A it occurs already during the first phase of the M-rise ( < 1 s). (ii) The introduction of a second mutation at the cytoplasmic surface in position 231 (helix G) restores wild type ground state absorption properties, kinetics of photocycle and of proton release, and uptake in the mutant R82A/G231C. In addition, kinetic H/D isotope effects provide evidence that the proton release mechanism in R82A/G231C and in wild type is similar. These results suggest the existence of long range interactions between the cytoplasmic and extracellular surface domains of bacteriorhodopsin mediated by salt bridges and hydrogen-bonded networks between helices C (Arg-82) and G (Asp-212 and Gly-231). Such long range interactions are expected to be of functional significance for activation and signal transduction in heptahelical Gprotein-coupled receptors.Allosteric interactions are well known and of great importance for water soluble enzymes, where ligand or substrate binding to a specific site alters the conformation and changes the affinity at a different site of the protein (for reviews see Refs. 1 and 2). For membrane-bound G-protein-coupled receptors analogous effects are expected to be of considerable functional significance. For this class of proteins ligand binding occurs mostly on the extracellular surface resulting in the active form of the receptor with binding and activation of the G-protein at the opposite cytoplasmic surface of the protein.The membrane protein bacteriorhodopsin (bR) 1 shares the heptahelical bundle motif with G-protein-coupled receptors. In this report, we present evidence for long range interactions between the extracellular and cytoplasmic parts of bR based on evidence from the mutant R82A and its second site revertant R82A/G231C.Bacteriorhodopsin acts as a light-driven proton pump in the plasma membrane of the archaebacterium Halobacterium salinarium. A retinylidene chromophore is bound via a protonated Schiff base linkage to Lys-216. Upon flash excitation bR undergoes a cyclic photoreaction with distinct spectroscopic intermediates and proton translocation from one side of the membrane to the other (for reviews see Refs. 3-5). In the first half of this photocycle, the Schiff base becomes deprotonated during the transition to the ...

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Section: Kinetics Of Proton Release and Uptakementioning

confidence: 57%

“…In R82A an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake, detected with pH indicator dyes in the aqueous solution, were already observed (20,21). Moreover, in R82A the pK a of Asp-85, which controls the purple to blue transition, is increased by about 5 pH units compared with wild type (wt) (22).…”

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confidence: 95%

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

Alexiev¹,

Mollaaghababa²,

Khorana³

et al. 2000

Journal of Biological Chemistry

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“…It has been observed that modification of protein groups in the vicinity of the retinal Schiff base shifts considerably the bR absorption spectrum [69] and affects drastically the rates of both thermally- [70] and photo- [71,72] [75,76,77]. At present, there exist a number of ab initio as well as semi-empirical techniques which try to account for the realistic atomic structure and charge distribution of the environment via explicit incorporation of protein (or solution) point charges in the electronic Hamiltonian of the quantum chemically treated substrate [78,79,80,81,82,83].…”

Section: Quantum Chemistry Of In Situ Retinalmentioning

confidence: 99%

“…It has been observed that modification of protein groups in the vicinity of the retinal Schiff base shifts considerably the bR absorption spectrum [69] and affects drastically the rates of both thermally- [70] and photo- [71,72] activated isomerization processes in bR. The experiments imply that the protein environment plays a crucial role in determining the physico-chemical properties of…”

Section: Quantum Chemistry Of In Situ Retinalmentioning

confidence: 99%

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Quantum Chemistry of in situ Retinal: Study of the Spectral Properties and Dark Adaptation of Bacteriorhodopsin

Logunov

Schulten

1996

Quantum Mechanical Simulation Methods for Studying Biological Systems

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1A combination of molecular dynamics and quantum chemistry techniques have been employed to study the electronic excitation and conformational potential surface of retinal in the binding site of bacteriorhodopsin (bR). The CASSCF(6,9)/6-31G level of ab initio calculations (within Gaussian92) has been used for the treatment of both the ground (S0) and excited (S1) states of retinal. Charges of all atoms in the protein are represented by spherical Gaussians and explicitly included in the electronic Hamiltonian of retinal. Spectral properties have been analyzed for the native bR pigment as well as for its D85N mutant.The calculated relative shift in the absorption maxima between the two pigments is in better agreement with experiment than the computed absolute parameters of the absorption line shapes. The dark adaptation processes in bacteriorhodopsin (which involves rotation around the 13-14 and the 15-N retinal double bonds) has been modelled by following the pre-defined reaction coordinate. Our simulations support the notion that the isomerization process is catalyzed by the protonation of an aspartic acid (Asp85) side group of bacteriorhodopsin. 2 IntroductionBacteriorhodopsin (bR) spans the cell membrane of Halobacterium halobium and functions as a light-driven proton pump. bR contains seven α-helices which enclose the prosthetic group, all-trans retinal, bound via a protonated Schiff base linkage to Lys-216. Figure 1a shows the chemical structure of retinal and its conventional numbering scheme. The bR structure is presented in Fig. 2a. Retinal absorbs light and undergoes a photoisomerization process; the thermal reversal of this reaction is coupled to transfer of a proton from the cytoplasmic side (top in Fig. 2a) to the extracellular side (bottom in Fig. 2a) of the protein.Recent reviews that discuss the structure and function of bR are [1,2,3,4,5,6,7]. Figure 1 here Bacteriorhodopsin accomplishes its function through a cyclic process initiated by absorption of a photon. This photon triggers an isomerization of retinal, which proceeds then through several intermediate states identified by their absorption spectra. An accepted kinetic scheme for this cycle is an unbranched series of intermediates, shown in Fig. 3. Photoisomerization occurs in the bR 568 → J 625 transition. During the L 550 → M 412 transition the Schiff base proton is transferred to Asp-85 and, subsequently, to the outside of the cell [8,9,10,11,12,13]. During the M 412 → N 520 transition, a proton is transferred to the Schiff base from Asp-96, which then takes up a proton from the cytoplasmic environment [13]. The reaction cycle is completed as the protein returns to bR 568 via the O 640 intermediate. Figure 2 here When bR is allowed to equilibrate in the dark, it converts within an hour to a 2:1 mixture containing 13-cis retinal and all-trans retinal bR [14]. The protein containing the 13-cis retinal isomer of bRis referred to as the dark adapted (DA) pigment of bR, bR 548 , which absorbs at 548 nm. bR 548 contains, actually, retinal in a 1...

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Visual Pigments as Photoreceptors

Kumauchi

Ebrey

2005

Handbook of Photosensory Receptors

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Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle

Cited by 179 publications

References 58 publications

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

Quantum Chemistry of in situ Retinal: Study of the Spectral Properties and Dark Adaptation of Bacteriorhodopsin

Visual Pigments as Photoreceptors

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