Directed evolution of bacteriorhodopsin for applications in bioelectronics

Wagner, Nicole L.; Greco, Jordan A.; Ranaghan, Matthew J.; Birge, Robert R.

doi:10.1098/rsif.2013.0197

Cited by 70 publications

(94 citation statements)

References 123 publications

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“…One functionally diverse class of light-activated proteins -opsins -occurs throughout the microbial world, where they facilitate phototactic and photophobic responses in algae [9,10], enhanced survival in response to nutrient starvation in bacteria [11], and conversion of solar energy into electrochemical energy in diverse microbes [12,13]. Knowing the molecular determinants for these proteins' light absorption properties will help us to understand their biological functions as well as to engineer new versions for applications in bioenergy [13], biomaterials [14], optogenetics [15], and live-cell imaging [16].…”

Section: Introductionmentioning

confidence: 99%

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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Section: Introductionmentioning

confidence: 99%

Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties

Engqvist

McIsaac

Dollinger

et al. 2015

Journal of Molecular Biology

124

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“…Site-saturation mutagenesis of Mero-1 at position G61 (selected from a marginal hit in the error-prone PCR library) led to Mero-2, with mutations P60S and G61L (Figure 2). Mutations that increase fluorescent brightness of microbial rhodopsins (Engqvist et al, 2014; Hochbaum et al, 2014; McIsaac et al, 2014) and increase occupancy of associated states in the photocycle (Maclaurin et al, 2013; Wagner et al, 2013) are known to be located proximal to ATR or the Schiff base. Thus, to guide further evolution, we generated a homology model of merocyanine-bound wild-type Arch based on the known structure of Archaerhodopsin-2 (86% amino acid identity; Figure 3) (Kouyama et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore

Herwig

Rice

Bedbrook

et al. 2017

Cell Chemical Biology

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Summary By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch-complex with a 200 nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.

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“…Over the last two decades, some potential applications of both mutants and wildtype bR have been proposed ranging from medicine to electronics [35][36][37][38][39][40]. On the other hand, bR can be interfaced with various nanomaterials for fabrication of highly efficient devices and advanced functional materials.…”

Section: Accepted Manuscriptmentioning

confidence: 99%