The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane

Kikukawa, Takashi; Araiso, Tsunehisa; Mukasa, K.; Shimozawa, Tateo; Kamo, Naoki

doi:10.1016/0014-5793(95)01408-x

Cited by 4 publications

(2 citation statements)

References 24 publications

(33 reference statements)

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“…The M intermediate was assigned by comparing the 13 C chemical shift value of 20- 13 C and 14- 13 C with those previously reported for the M-intermediate [ 31 , 37 ] ( Table S1 ). It has been reported that an N intermediate is not observed in D96N-BR, because the proton donor D96 is replaced by N96 [ 22 , 23 , 24 , 25 ]. Therefore, another peak was assigned to the L intermediate and the chemical shifts agree with previously reported values [ 31 ] ( Table S1 ).…”

Section: Resultsmentioning

confidence: 99%

“…In the photoreaction cycle shown in Figure 1 B, the photoreaction L ↔ M refers to proton exchange between SB and Asp85, while M ↔ N refers to proton exchange between Asp96 and SB. Therefore, replacement of Asp96 with Asn96 (D96N-BR) prevents the accumulation of any intermediate subsequent to the M intermediates, such as the N and O intermediates, while retaining proton pump activity [ 22 , 23 , 24 , 25 ]. Thus, the half-lives of the M and L intermediates are significantly increased in the D96N-BR mutant.…”

Section: Introductionmentioning

confidence: 99%

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Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR

et al. 2022

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Bacteriorhodopsin (BR) functions as a light-driven proton pump that transitions between different states during the photocycle, such as all-trans (AT; BR568) and 13-cis, 15-syn (CS; BR548) state and K, L, M1, M2, N, and O intermediates. In this study, we used in situ photoirradiation 13C solid-state NMR to observe a variety of photo-intermediates and photoreaction pathways in [20-13C]retinal-WT-BR and its mutant [20-13C, 14-13C]retinal-D96N-BR. In WT-BR, the CS state converted to the CS* intermediate under photoirradiation with green light at −20 °C and consequently converted to the AT state in the dark. The AT state converted to the N intermediate under irradiation with green light. In D96N-BR, the CS state was converted to the CS* intermediate at −30 °C and consequently converted to the AT state. Simultaneously, the AT state converted to the M and L intermediates under green light illumination at −30 °C and subsequently converted to the AT state in the dark. The M intermediate was directly excited to the AT state by UV light illumination. We demonstrated that short-lived photo-intermediates could be observed in a stationary state using in situ photoirradiation solid-state NMR spectroscopy for WT-BR and D96N-BR, enabling insight into the light-driven proton pump activity of BR.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR

et al. 2022

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Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol

Kikukawa¹,

Araiso²,

Shimozawa³

et al. 1997

Biophysical Journal

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The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol. In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay. This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension. In the presence of ethanol, however, we observed the wavelength-dependent anisotropies. The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates. The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin. The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.

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Reorientational Motions of the D96N and T46V/D96N Mutants of Bacteriorhodopsin in the Purple Membrane

Harms

Johnson

1997

Photochem & Photobiology

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Abstract— The reorientational motions of the D96N and T46V/D96N mutants of bacteriorhodopsin in purple membrane have been investigated by time‐resolved linear dichroism measurements. The reorientations in the early stages of the photocycle are identical to those observed in wild‐type bacteriorhodopsin: anisotropics of photocycle intermediates in both D96N and T46V/D96N are rK= 0.38±0.01, rL= 0.35±0.01, rM= 0.35±0.01. The anisotropy of the initial state, rBR, exhibits decays to zero in D96N and to negative values in T46V/D96N on the time scale of tens of milliseconds. This anisotropy decay can be explained by a model that involves the motion of unexcited or spectator proteins adjacent to a photocycling protein. The amplitude and time constants of spectator reorientational motion are similar to those that have been observed in the wild type. Contributions from the anisotropy of the N‐state were detected in measurements of the T46V/D96N mutant, in which a large N‐state population accumulates. The value of rN is estimated to be 0.30±0.05 in T46V/D96N.

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The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane

Cited by 4 publications

References 24 publications

Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR

Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol

Reorientational Motions of the D96N and T46V/D96N Mutants of Bacteriorhodopsin in the Purple Membrane

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