Tuning the Electronic Absorption of Protein-Embedded All-
            <i>trans</i>
            -Retinal

Wang, Wenjing; Nossoni, Z.; Berbasova, Tetyana; Watson, Camille T.; Yapici, Ipek; Lee, Kin Sing Stephen; Vasileiou, Chrysoula; Geiger, James H.; Borhan, Babak

doi:10.1126/science.1226135

Cited by 115 publications

(196 citation statements)

References 27 publications

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“…Clearly, bacterioruberin is absent in the bAR membrane and the UV-VIS spectrum is similar to the bacterioruberin-excluded AR4-xz515, as reported before [18]. A small blue shift in bAR when compared with BR may indicate that a slightly different protein packing, which may affect the retinal binding pocket in bAR [53], and hydrophobic binding pocket in rhodopsin has also been shown to be functionally important [54]. It is also worth noticing that most members of the Halobacteriaceae, including H. salinarum, possess bacterioruberin [55,56], whereas no bacterioruberin is present in the purple membrane of H. salinarum.…”

Section: Assessments Of Recombinant Ar4supporting

confidence: 57%

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

Cao

Ding

Peng

et al. 2015

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Archaerhodopsin 4 (AR4), a new member of the microbial rhodopsin family, is isolated from Halobacterium species xz515 in a Tibetan salt lake. AR4 functions as a proton pump similar to bacteriorhodopsin (BR) but with an opposite temporal order of proton uptake and release at neutral pH. However, further studies to elucidate the mechanism of the proton pump and photocycle of AR4 have been inhibited due to the difficulty of establishing a suitable system in which to express recombinant AR4 mutants. In this paper, we report a reliable method for expressing recombinant AR4 in Halobacterium salinarum L33 with a high yield of up to 20mg/l. Experimental results show that the recombinant AR4 retains the light-driven proton pump characteristics and photo-cycling kinetics, similar to that in the native membrane. The functional role of bacterioruberin in AR4 and the trimeric packing of AR4 in its native and recombinant forms are investigated through light-induced kinetic measurements, two-dimensional solid-state NMR experiments, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). Such approaches provide new insights into structure-function relationships of AR4, and form a basis for other archaeal rhodopsins.

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Section: Assessments Of Recombinant Ar4supporting

confidence: 57%

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

Cao

Ding

Peng

et al. 2015

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…The stabilization and destabilization of the S0 and S1 states is in turn governed by interactions between retinal and the protein binding pocket. Four factors have been reported to affect the S0-S1 energy gap: (i) conjugation of the retinal chromophore [26,34], (ii) polarity of the retinal-binding cavity [35,36], (iii) chromophore positioning effects [37], and (iv) interaction between Schiff base and the Schiff base counterion [38,39]. In our dataset, blue-shifting mutations are correlated with changes in polarity along the retinal molecule ( Figure 4).…”

Section: Routes To Spectral Tuning In Opsinsmentioning

confidence: 78%

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Engqvist

McIsaac

Dollinger

et al. 2015

Journal of Molecular Biology

124

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Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for lightharvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal binding pocket of Gloeobacter violacaeus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to +/-80 nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurrred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while redshifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty-four of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the wild-type protein.

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“…Color tuning has been studied with fluorescent proteins (71), PYP (72), and retinal proteins ("opsin shift") (73), which all adjust the energy gaps between the excited and ground states. These energy gaps correlate well with electrostatic interactions experienced by the chromophores (74)(75)(76).…”

Section: Resultsmentioning

confidence: 99%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.split GFP | potential energy surface | photodissociation | cis-trans isomerization | photosensory protein O ptical techniques for investigating biological processes in vivo can achieve subcellular spatial and millisecond temporal resolution by using genetically encoded light-responsive proteins (1). Photoactivatable proteins are used as either imaging tools, such as reversibly photoswitchable fluorescent proteins (RSFPs), with fluorescence that can be modulated by light (2) or optogenetic switches that convert light input into physiological outputs, such as channelrhodopsins, which can regulate ion flow through membranes in response to light (3). Split GFPs have been widely applied for imaging as fluorescent reporters of cellular processes, because they are small (∼25 kDa), are stable in cytosol, produce chromophores autocatalytically, and are amenable to mutation and circular permutation (4). Typically, the protein is expressed as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another, offering readouts of protein-protein colocalizaton with low background and high specificity (5). However, this technique can generate misleading results, because the GFP complexes, after being formed and fluorescing, are bound irreversibly, which can be highly perturbative to the processes being studied, especially if the protein interaction being probed is ordinarily reversible (6). Although split GFP complexes essentially do not dissocia...

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Tuning the Electronic Absorption of Protein-Embedded All- trans -Retinal

Cited by 115 publications

References 27 publications

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

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