Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

Glantz, Spencer T.; Berlew, Erin E.; Jaber, Zaynab; Schuster, Benjamin S.; Gardner, Kevin H.; Chow, Brian Y.

doi:10.1073/pnas.1802832115

Cited by 54 publications

(103 citation statements)

References 79 publications

(110 reference statements)

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“…65,68 In general, for different LOV receptors these initial light-induced conformational changes consistently culminate in a destabilization of the interaction between the outer face of the β-sheet and N-and C-terminal ancillary elements packed against it, often causing their detachment. This is most prominently evidenced in the dissociation and unfolding of the Jα helix in phototropin LOV sensors 57 but is also reflected in the DNA-binding LOV receptors aureochromes 69,70 and EL222, 71 Neurospora crassa Vivid, 39,67 RGS-LOV proteins, 72 a recently discovered RNAbinding LOV receptor, 38 and in the monomeric LOV sensor histidine kinase EL346. 6 Applied to YF1 as a paradigm for the more prevalent dimeric SHKs, the following scenario emerges.…”

Section: Lov Signalingmentioning

confidence: 99%

Signal transduction in photoreceptor histidine kinases

Möglich

2019

Protein Science

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Two‐component systems (TCS) constitute the predominant means by which prokaryotes read out and adapt to their environment. Canonical TCSs comprise a sensor histidine kinase (SHK), usually a transmembrane receptor, and a response regulator (RR). In signal‐dependent manner, the SHK autophosphorylates and in turn transfers the phosphoryl group to the RR which then elicits downstream responses, often in form of altered gene expression. SHKs also catalyze the hydrolysis of the phospho‐RR, hence, tightly adjusting the overall degree of RR phosphorylation. Photoreceptor histidine kinases are a subset of mostly soluble, cytosolic SHKs that sense light in the near‐ultraviolet to near‐infrared spectral range. Owing to their experimental tractability, photoreceptor histidine kinases serve as paradigms and provide unusually detailed molecular insight into signal detection, decoding, and regulation of SHK activity. The synthesis of recent results on receptors with light‐oxygen‐voltage, bacteriophytochrome and microbial rhodopsin sensor units identifies recurring, joint signaling strategies. Light signals are initially absorbed by the sensor module and converted into subtle rearrangements of α helices, mostly through pivoting and rotation. These conformational transitions propagate through parallel coiled‐coil linkers to the effector unit as changes in left‐handed superhelical winding. Within the effector, subtle conformations are triggered that modulate the solvent accessibility of residues engaged in the kinase and phosphatase activities. Taken together, a consistent view of the entire trajectory from signal detection to regulation of output emerges. The underlying allosteric mechanisms could widely apply to TCS signaling in general.

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Section: Lov Signalingmentioning

confidence: 99%

Signal transduction in photoreceptor histidine kinases

Möglich

2019

Protein Science

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“…Quorum sensing experiments were performed as described previously 1 . Recombinant EL222 was bacterially produced and FPLC-purified as described previously 21 . Fluorescence detection limit was measured by reported methods for determining plate reader sensitivity 22 , with 0.3 µM NIST-traceable fluorescein and an integration time of 1.25 seconds.…”

Section: Methodsmentioning

confidence: 99%

An Open-Source Plate Reader

Szymula

Magaraci

Patterson

et al. 2018

Preprint

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Microplate readers are foundational instruments in experimental biology and bioengineering that enable multiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construction, validation, and benchmarking of an opensource microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs <$3500, a fraction of the cost of commercial plate readers, and can detect the fluorescence of common dyes down to ~10 nanomolar concentration. Functional capabilities were demonstrated in context of synthetic biology, optogenetics, and photosensory biology: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing, and by flavin photocycling kinetic measurements of a LOV (light-oxygen-voltage) domain photoreceptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and automating it using the custom Python-based API (Application Program Interface) are provided. This work contributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid prototyping capabilities and lowcost electronics, optoelectronics, and microcomputers.

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“…Fluorescence imaging of water-in-oil (w/o) emulsions with lipid-stabilized droplet interfaces offers a complementary platform for screening protein-lipid interactions, determining relative affinities, and monitoring interaction dynamics (Glantz et al, 2018). In this system, the droplet interior emulates the cytosol, while the lipid monolayer-stabilized droplet interface emulates the plasma membrane inner leaflet (Figure 1).…”

Section: Overviewmentioning

confidence: 99%

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions

Glantz¹,

Berlew²,

Chow³

2019

Methods in Enzymology

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The ability to rapidly screen interactions between proteins and membrane-like interfaces would aid in establishing the structure-function of protein-lipid interactions, provide a platform for engineering lipid-interacting protein tools, and potentially inform the signaling mechanisms and dynamics of membrane-associated proteins. Here, we describe the preparation and application of water-in-oil (w/o) emulsions with lipid-stabilized droplet interfaces that emulate the plasma membrane inner leaflet with tunable composition. Fluorescently labeled proteins are easily visualized in these synthetic cell-like droplets on an automated inverted fluorescence microscope, thus allowing for both rapid screening of relative binding and spatiotemporally resolved analyses of for example, protein-interface association and dissociation dynamics and competitive interactions, using commonplace instrumentation. We provide protocols for droplet formation, automated imaging assays and analysis, and the production of the positive control protein BcLOV4, a natural photoreceptor with a directly light-regulated interaction with anionic membrane phospholipids that is useful for optogenetic membrane recruitment.

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Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

Cited by 54 publications

References 79 publications

Signal transduction in photoreceptor histidine kinases

Signal transduction in photoreceptor histidine kinases

An Open-Source Plate Reader

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions

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