Biosensors based on peptide exposure show single molecule conformations in live cells

Liu, Bei; Stone, Orrin J.; Pablo, Michael; Herron, J. Cody; Nogueira, Ana T.; Dağliyan, Onur; Grimm, Jonathan B.; Lavis, Luke D.; Elston, Timothy C.; Hahn, Klaus M.

doi:10.1016/j.cell.2021.09.026

Cited by 22 publications

(16 citation statements)

References 103 publications

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“…774,790 Another conformation-sensitive fluorescence method has been recently published: it exploits the conditional recognition of a peptide-tag by a fluorescent binder, which occurs upon a conformational opening of the studied protein. 796 To this aim, i) a chimera is designed to keep the peptide-tag non-accessible as long as the studied protein does not adopt an open conformation, and ii) a fluorescent binder is selected that binds the tag selectively but with a moderate affinity. Combined with TIRF and single-particle tracking, it permitted to characterize the conformational exchange of the active (open) and inactive (close) forms of the kinase Src at the membrane of transfected mammalian cells.…”

Section: In-cell Fret Microscopymentioning

confidence: 99%

“…Another conformation-sensitive fluorescence method has been recently published. It exploits the conditional recognition of a peptide-tag by a fluorescent binder, which occurs upon a conformational opening of the studied protein . To this aim, (i) a chimera is designed to keep the peptide-tag nonaccessible as long as the studied protein does not adopt an open conformation and (ii) a fluorescent binder is selected that binds the tag selectively but with a moderate affinity.…”

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

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In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Theillet

2022

Chem. Rev.

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In-cell structural biology aims at extracting structural information about proteins or nucleic acids in their native, cellular environment. This emerging field holds great promises and is already providing new facts and outlooks of interest at both fundamental and applied levels. NMR spectroscopy has important contributions on this stage: it brings information on a broad variety of nuclei at the atomic scale, which ensures its great versatility and uniqueness. Here, we detail the methods, the fundamental knowledge and the applications in biomedical engineering related to in-cell NMR. We finally propose a brief overview of the main other techniques in the field (EPR, smFRET, cryo-ET…) to draw some advisable developments for in-cell NMR. In the era of large-scale screenings and deep learning, both accurate and qualitative experimental evidences are as essential as ever to understand the interior life of cells. In-cell structural biology by NMR spectroscopy can generate such a knowledge, and it does so at the atomic scale. This review is meant to deliver comprehensive but accessible information, with advanced technical details and reflections on the methods, the nature of the results and the future of the field. CONTENTS 5.1.1. In-cell EPR 5.1.2. In-cell FRET microscopy 5.1.3. Mass-spectrometry 5.1.4. Cryo-ET 5.2. The specific benefits of in-cell NMR 5.3. The future technical challenges of in-cell NMR 6. Conclusion Author Information

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Section: In-cell Fret Microscopymentioning

confidence: 99%

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Theillet

2022

Chem. Rev.

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“…Cdc42 is a regulator of the actin cytoskeleton, so we next examined its localization during frustrated phagocytosis. We recently developed tools for observing and analyzing single molecule conformational changes in living cells [ 42 ]. We made use of these techniques to visualize Cdc42 during frustrated phagocytosis (Figs 1D–1G and S1C ).…”

Section: Resultsmentioning

confidence: 99%

Spatial models of pattern formation during phagocytosis

Herron

Liu

et al. 2022

PLoS Comput Biol

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Phagocytosis, the biological process in which cells ingest large particles such as bacteria, is a key component of the innate immune response. Fcγ receptor (FcγR)-mediated phagocytosis is initiated when these receptors are activated after binding immunoglobulin G (IgG). Receptor activation initiates a signaling cascade that leads to the formation of the phagocytic cup and culminates with ingestion of the foreign particle. In the experimental system termed “frustrated phagocytosis”, cells attempt to internalize micropatterned disks of IgG. Cells that engage in frustrated phagocytosis form “rosettes” of actin-enriched structures called podosomes around the IgG disk. The mechanism that generates the rosette pattern is unknown. We present data that supports the involvement of Cdc42, a member of the Rho family of GTPases, in pattern formation. Cdc42 acts downstream of receptor activation, upstream of actin polymerization, and is known to play a role in polarity establishment. Reaction-diffusion models for GTPase spatiotemporal dynamics exist. We demonstrate how the addition of negative feedback and minor changes to these models can generate the experimentally observed rosette pattern of podosomes. We show that this pattern formation can occur through two general mechanisms. In the first mechanism, an intermediate species forms a ring of high activity around the IgG disk, which then promotes rosette organization. The second mechanism does not require initial ring formation but relies on spatial gradients of intermediate chemical species that are selectively activated over the IgG patch. Finally, we analyze the models to suggest experiments to test their validity.

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“…A recently described Src biosensor is based on intermolecular FRET using a 7-mer peptide tag that is only exposed when Src is activated. When exposed, it can bind a reporter protein bringing a donor–acceptor fluorescent pair to a short distance [ 35 ]. However, these biosensors monitor the formation of a well-defined complex around the globular domains.…”

Section: Discussionmentioning

confidence: 99%

A FRET-Based Biosensor for the Src N-Terminal Regulatory Element

et al. 2022

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In signaling proteins, intrinsically disordered regions often represent regulatory elements, which are sensitive to environmental effects, ligand binding, and post-translational modifications. The conformational space sampled by disordered regions can be affected by environmental stimuli and these changes trigger, vis a vis effector domain, downstream processes. The disordered nature of these regulatory elements enables signal integration and graded responses but prevents the application of classical approaches for drug screening based on the existence of a fixed three-dimensional structure. We have designed a genetically encodable biosensor for the N-terminal regulatory element of the c-Src kinase, the first discovered protooncogene and lead representative of the Src family of kinases. The biosensor is formed by two fluorescent proteins forming a FRET pair fused at the two extremes of a construct including the SH4, unique and SH3 domains of Src. An internal control is provided by an engineered proteolytic site allowing the generation of an identical mixture of the disconnected fluorophores. We show FRET variations induced by ligand binding. The biosensor has been used for a high-throughput screening of a library of 1669 compounds with seven hits confirmed by NMR.

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Biosensors based on peptide exposure show single molecule conformations in live cells

Cited by 22 publications

References 103 publications

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Spatial models of pattern formation during phagocytosis

A FRET-Based Biosensor for the Src N-Terminal Regulatory Element

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