Comparison of the Dynamics of the Primary Events of Bacteriorhodopsin in Its Trimeric and Monomeric States

Wang, Jianping; Link, Stephan; Heyes, Colin D.; El‐Sayed, Mostafa A.

doi:10.1016/s0006-3495(02)73925-8

Cited by 55 publications

(88 citation statements)

References 73 publications

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“…The light-dark adaptation yielded a 5-nm shift with a dark absorption maxima of 549 nm and light absorption maxima of 554 nm. These results indicated that the majority of active bR is likely in a monomeric form in the presence of NLPs formed around a DMPC bilayer (26,27). This is in agreement with other studies that used prepurified apolipoprotein scaffolds to solubilize native forms of bR (2).…”

Section: Resultssupporting

confidence: 91%

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Cappuccio

Blanchette

Sulchek

et al. 2008

Molecular & Cellular Proteomics

112

123

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Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cellfree extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein ⌬1-49 apolipoprotein A-I fragment (⌬49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR 3 M transition. Importantly the functional bR was solubilized in discoidal bR⅐NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with ⌬49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

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Section: Resultssupporting

confidence: 91%

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Cappuccio

Blanchette

Sulchek

et al. 2008

Molecular & Cellular Proteomics

112

123

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“…Similar to BR, this most likely reflects a partial isomerization of the retinal to a 13- cis /C=N syn configuration 35 . Incorporation of AR3 in detergent micelles formed from OG or dodecylmaltoside (DM) also resulted in a distinct blue-shift of the λ max to near 560nm even after light-adaptation and most likely reflects an increased 13-cis retinal content of the protein which is associated with a monomer relative to trimer organization of the protein 36,37 . For example, a downshifted absorbance of both light-and dark-adapted BR to 554 and 549 nm, respectively, is observed in BR incorporated into nanolipoprotein particles (NLPs) containing dimyristoylphosphatidylcholine (DMPC) lipids 38 .…”

Section: Resultsmentioning

confidence: 99%

Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

et al. 2012

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Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3–11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (Oall-trans) is found. At higher pH a second 13-cis retinal N-like species (N13-cis) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N13-cis detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N13-cis to M13-cis levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.

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“…Purple membrane was prepared from Halobacterium salinarum S9 according to a previous method. 42 Monomeric bR from H. salinarum, named HsbR, was prepared by solubilizing purple membrane (PM) suspension in Triton X-100 based on the method by Wang et al 43 PM suspension was mixed with Triton X-100 at a ratio 1:7 (w/w) and kept in the dark for more than 48 h. The HsbR monomer in Triton X-100 was further extracted after 10 min of centrifugation at 18 407g and slight sediment was discarded. HsbR monomer solution was prepared in 25 mM phosphate buffer solution at pH ∼7.0, and the UV−vis absorption spectrum maximum accordingly shifted from 568 to 553 nm.…”

Section: Methodsmentioning

confidence: 99%

Photochemistry of a Dual-Bacteriorhodopsin System in Haloarcula marismortui: HmbRI and HmbRII

Tsai

Yang

et al. 2014

J. Phys. Chem. B

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Recently, a dual-bacteriorhodopsin system, containing HmbRI and HmbRII, has been found in Haloarcula marismortui (Mol. Microbiol. 2013, 88, 551-561), and the light-driven proton pump activities were intrinsically different in a wide pH range. Compared with bacteriorhodopsin in H. salinarum (HsbR), the identical steady-state absorption contours of HsbR and HmbRs in the visible range indicated similarities in the retinal pocket. In addition, other reactive residues, including the proton relay channel, proton release group, and proton collecting funnel at the cytoplasm, were mostly conserved. We employed transient absorption spectroscopy and global analysis to characterize the photocycle intermediates and kinetics of HmbRI and HmbRII in the pH range of 4-8. The features of the time-resolved difference spectra of HmbRI indicated that the photocycle of HmbRI mainly followed the conventional pathway, including intermediates M, N, and O. A minute bypassed pathway from intermediate M needed to be included to better match the experimental data. The corresponding intermediate M' is attributed to the all-trans deprotonated Schiff base retinal, indicating the occurrence of retinal reisomerization prior to the reprotonation of the deprotonated Schiff base following the decay of intermediate M. Regarding HmbRII, its photocycle only followed the intermediates M and N, without intermediate O. The plausible molecular mechanisms, including the effects of the lengths of the loops and the distribution of the charged residues in the bacterio-opsin interior, were proposed to explain the differences in the photocycles. The pH-dependent photocycles were also investigated, and the results supported our proposed mechanism. Unravelling the photocycles of the HmbRs in the Haloarcula marismortui provided evidence that not only expanding the functional pH ranges but also the turnover kinetics are the strategies of the dual-bR system in the evolution of microbes in extreme environments.

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Comparison of the Dynamics of the Primary Events of Bacteriorhodopsin in Its Trimeric and Monomeric States

Cited by 55 publications

References 73 publications

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

Photochemistry of a Dual-Bacteriorhodopsin System in Haloarcula marismortui: HmbRI and HmbRII

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