Asp 46 can substitute for Asp 96 as the Schiff base proton donor in bacteriorhodopsin

Coleman, Matthew A.; Nilsson, Anders; Russell, Terence S.; Rath, Parshuram; Pandey, Rashmi; Rothschild, Kenneth J.

doi:10.1021/bi00047a027

Cited by 7 publications

(7 citation statements)

References 48 publications

(108 reference statements)

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“…This frequency is typical for the CdO stretch mode of Asn and may reflect a change in hydrogen bonding of Asn76 during the SR 587 f S 373 transition due to Schiff base deprotonation. A similar band has been observed when Asn is substituted for Asp96 and Thr46 in the BR f M difference spectrum (Gerwert et al, 1989;Coleman et al, 1995).…”

Section: Resultssupporting

confidence: 75%

Asp76 Is the Schiff Base Counterion and Proton Acceptor in the Proton-Translocating Form of Sensory Rhodopsin I

et al. 1996

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Both sensory rhodopsin I, a phototaxis receptor, and bacteriorhodopsin, a light-driven proton pump, have homologous residues which have been identified as critical for bacteriorhodopsin functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions as both the Schiff base counterion and the proton acceptor. Sensory rhodopsin I exists in a pH dependent equilibrium between two different forms in the absence of its transducer protein HtrI. At pH below 7, it exists primarily in a blue form (lambda max = 587 nm) which functions as a phototaxis signal transducer when complexed to HtrI, while at higher pH, it converts to a purple proton-transporting form similar to bacteriorhodopsin (lambda max = 550 nm). We report ATR-FTIR difference spectra obtained from both low- and high-pH forms of purified sensory rhodopsin I reconstituted into lipid vesicles. The low-pH species has an ethylenic C = C stretch mode at 1520 cm-1 which shifts to 1526 cm-1 in the high-pH form. No frequency shift was found for the mutant D76N, in agreement with visible absorption measurements. Weak negative/positive bands at 1763/1751 cm-1 previously assigned to a perturbation of the C = O stretch mode of Asp76 during S373 formation in the low-pH form are replaced by a single intense positive band near 1749 cm-1 in the high-pH form. These results along with the effects of H/D exchange show that Asp76 is protonated in the signal-transducing form of sensory rhodopsin I and is ionized and functions as the counterion and Schiff base proton acceptor in the proton-transporting high-pH form of sensory rhodopsin I similar to bacteriorhodopsin.

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Section: Resultssupporting

confidence: 75%

Asp76 Is the Schiff Base Counterion and Proton Acceptor in the Proton-Translocating Form of Sensory Rhodopsin I

et al. 1996

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“…Therefore, we conclude that the time constants characterizing the M-decay of all but one sample (D38C) are increased slightly after the exchange of the natural amino acids by cysteines. This may be due to the charge changes on the cytoplasmic surface of the BR molecule or, as in the case of T46C, changes in the hydrogen network or a steric modification in the interior of the protein (Rothschild et al, 1992;Coleman et al, 1995;LeCoutre et al, 1996). The most dramatic change is found for D38C, in which the photocycle is retarded close to values known from D96 mutants (Riesle et al, 1996).…”

Section: Biophysical Journalmentioning

confidence: 89%

“…1 b). Several studies indicate that T46 interacts with D96 (Marti et al, 1991;Rothschild et al, 1992;Coleman et al, 1995) and may be part of a hydrogen network between the cytoplasmic surface and the Schiff base (LeCoutre et al, 1996). The mutation at position 46 may thus influence the accessibility of D96 for the proton delivered by the cytoplasmic medium, which may result in the observed delay of the N decay.…”

Section: Discussionmentioning

confidence: 99%

Spin-labeling studies of the conformational changes in the vicinity of D36, D38, T46, and E161 of bacteriorhodopsin during the photocycle

Rink

Riesle

Oesterhelt

et al. 1997

Biophysical Journal

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Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin-labeled bacteriorhodopsin mutants is used to study structural changes during the photocycle. After exchange of the native amino acids D36 and D38 in the A-B loop, E161 in the E-F loop, and T46 in the putative proton channel by cysteines, these positions were modified by a methanethiosulfonate spin label. Time-resolved EPR spectroscopy reveals spectral changes during the photocycle for the mutants with spin labels attached to C36, C161, and C46. A comparison of the transient spectral amplitudes with simulated EPR difference spectra shows that the detected signals are due to changes in the spin label mobility and not to possible polarity changes in the vicinity of the attached spin label. The kinetic analysis of the EPR and the visible data with a global fitting procedure exhibits a structural rearrangement near position 161 in the E-F loop in the M state. The environmental changes at positions 36 and 46, however, occur during the M-to-N transition. All structural changes reverse with the recovery of the BR ground state. No structural changes are detected with a spin label attached to C38.

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“…Mutants of bacteriorhodopsin were expressed in H. salinarium. All methods and procedures were previously described [27,28]. Wild type (WT) and mutant strains were grown, and the purple membrane was isolated, using previously described procedures [29].…”

Section: Expression Of T89n T46n T46n/t89nmentioning

confidence: 99%

Detection of threonine structural changes upon formation of the M-intermediate of bacteriorhodopsin: evidence for assignment to Thr-89

Liu

Lee

Coleman

et al. 1998

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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The behavior of threonine residues in the bacteriorhodopsin (bR) photocycle has been investigated by Fourier transform infrared difference spectroscopy. L-Threonine labeled at the hydroxyl group with 18O (L-[3-(18)O]threonine) was incorporated into bR and the bR-->M FTIR difference spectra measured. Bands are assigned to threonine vibrational modes on the basis of 18O induced isotope frequency shifts and normal mode calculations. In the 3500 cm-1 region, a negative band is assigned to the OH stretch of threonine. In the 1125 cm-1 region, a negative band is assigned to a mixed CH3 rock/CO stretch mode. The frequency of both these bands indicates the presence of at least one hydrogen bonded threonine hydroxyl group in light adapted bR which undergoes a change in structure by formation of the M intermediate. Spectral changes induced by the substitution Thr-89-->Asn but not Thr-46-->Asn or Asp-96-->Asn are consistent with the assignment of these bands to Thr-89. These results along with another related study on the mutant Thr-89-->Asn indicate that the active site of bR includes Thr-89 and that its interaction with the retinylidene Schiff base and Asp-85 may play an important role in regulating the color of bacteriorhodopsin and the transfer of a proton to the Schiff base.

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Asp 46 can substitute for Asp 96 as the Schiff base proton donor in bacteriorhodopsin

Cited by 7 publications

References 48 publications

Asp76 Is the Schiff Base Counterion and Proton Acceptor in the Proton-Translocating Form of Sensory Rhodopsin I

Asp76 Is the Schiff Base Counterion and Proton Acceptor in the Proton-Translocating Form of Sensory Rhodopsin I

Spin-labeling studies of the conformational changes in the vicinity of D36, D38, T46, and E161 of bacteriorhodopsin during the photocycle

Detection of threonine structural changes upon formation of the M-intermediate of bacteriorhodopsin: evidence for assignment to Thr-89

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