Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks

Clair, Erica C. Saint; Ogren, John I.; Mamaev, Sergey; Kralj, Joel M.; Rothschild, Kenneth J.

doi:10.1007/s10867-011-9246-4

Cited by 17 publications

(23 citation statements)

References 66 publications

(121 reference statements)

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“…The methods for expression, purification, and reconstitution of WT AR3 in E. coli polar lipids is similar to a previous report 24 with some modifications. Unlike typical expression of BR or archaerhodopsins in halobacterial strains 13 , which endogenously express all- trans retinal, E. coli does not express retinal and thus expression of functional archaerhodopsin requires supplementation 16 .…”

Section: Methodsmentioning

confidence: 82%

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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Section: Methodsmentioning

confidence: 82%

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

et al. 2012

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“…(Adapted from [56].) Although, AR3 does not exhibit the same pattern of high frequency OH stretch bands seen in BR, this similarity indicates that there exists a conserved network of hydrogen bonds in both proteins which might serve as a proton wire [56].…”

Section: Structural Changes Of Internal Water Moleculesmentioning

confidence: 99%

“…In the case of BR, the detection of such broad absorbance bands has been associated with specific steps in proton transport [89,[93][94][95]. In a recent rapid-scan FTIR difference study of the light-driven proton pump from Halorubrum sodomense known as archaerhodopsins-3 broad continuum IR absorbance was discovered that is very similar to BR [56]. Fig.…”

Section: Structural Changes Of Internal Water Moleculesmentioning

confidence: 99%

“…These studies were all conducted in collaboration with John Spudich, a leader in the microbial rhodopsin field at the University of Texas Health Science Center at Houston. They included FTIR difference studies along with RRS on halorhodopsin (HR) [197], sensory rhodopsins (SRI, SRII) from archaebacterial [29,31,34,38,184], fungal neurospora rhodopsin (NO) [30], anabaena sensory rhodopsin from cyanobacteria (ASR) [32], green and blue proteorhodopsin proton pumps from marine bacteria (PRs) [4,5,24,28,33,129,130]; archaerhodopsins-3 (AR3), a BR-like protein [56,216] and most recently channelrhodopsins (ChRs) from algae [173,174,176,250]. These proteins span a range of functions which are representative of diverse biomembranes process and include active anion transport (HRs), signal transduction (SRI, SRII and ASR), and light-gated ion channels (ChRs).…”

Section: Beyond Bacteriorhodopsinmentioning

confidence: 99%

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The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Rothschild

2016

BSI

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Abstract. Membrane proteins facilitate some of the most important cellular processes including energy conversion, ion transport and signal transduction. While conventional infrared absorption provides information about membrane protein secondary structure, a major challenge is to develop a dynamic picture of the functioning of membrane proteins at the molecular level. The introduction of FTIR difference spectroscopy around 1980 to study structural changes in membrane proteins along with a number of associated techniques including protein isotope labeling, site-directed mutagenesis, polarization dichroism, attenuated total reflection and time-resolved spectroscopy have led to significant progress towards this goal. It is now possible to routinely detect conformational changes of individual amino acid residues, backbone peptides, binding ligands, chromophores and even internal water molecules under physiological conditions with time-resolution down to nanoseconds. The advent of ultrafast pulsed-IR lasers has pushed this time-resolution down to femtoseconds. The early development of FTIR difference spectroscopy as applied to membrane proteins with special focus on bacteriorhodopsin is reviewed from a personal perspective.

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“…The tilting of helix F has been further defined by EPR using dipolar coupling distance measurements [16–18] and by direct and dynamic visualization using high-speed AFM [19]. Elegant time-resolved molecular spectroscopic studies have identified also residue changes and water molecule movements in the E → C transition in BR [20–22], but to test the generality of the conformational change in the microbial rhodopsin family, the two well-established properties of the C conformer considered here are (i) the connection of the Schiff base to the cytoplasmic side of the protein and (ii) an open channel from the Schiff base to the cytoplasm, detectable structurally as a tilting of the cytoplasmic portion of helix F away from neighboring helices.…”

Section: The Ion Pumping Mechanismmentioning

confidence: 99%

Mechanism divergence in microbial rhodopsins

Spudich¹,

Sineshchekov²,

Govorunova³

2014

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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A fundamental design principle of microbial rhodopsins is that they share the same basic light-induced conversion between two conformers. Alternate access of the Schiff base to the outside and to the cytoplasm in the outwardly open “E” conformer and cytoplasmically open “C” conformer, respectively, combined with appropriate timing of pKa changes controlling Schiff base proton release and uptake make the proton path through the pumps vectorial. Phototaxis receptors in prokaryotes, sensory rhodopsins I and II, have evolved new chemical processes not found in their proton pump ancestors, to alter the consequences of the conformational change or modify the change itself. Like proton pumps, sensory rhodopsin II undergoes a photoinduced E → C transition, with the C conformer a transient intermediate in the photocycle. In contrast, one light-sensor (sensory rhodopsin I bound to its transducer HtrI) exists in the dark as the C conformer and undergoes a light-induced C → E transition, with the E conformer a transient photocycle intermediate. Current results indicate that algal phototaxis receptors channelrhodopsins undergo redirected Schiff base proton transfers and a modified E → C transition which, contrary to the proton pumps and other sensory rhodopsins, is not accompanied by the closure of the external half-channel. The article will review our current understanding of how the shared basic structure and chemistry of microbial rhodopsins have been modified during evolution to create diverse molecular functions: light-driven ion transport and photosensory signaling by protein-protein interaction and light-gated ion channel activity.

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Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks

Cited by 17 publications

References 66 publications

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

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

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Mechanism divergence in microbial rhodopsins

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