Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

Gruber, Simon; Cornea, Rǎzvan L.; Peterson, Kurt C.; Gillispie, Gregory D.; Robia, Seth L.; Thomas, David D.

doi:10.1016/j.bpj.2013.11.2409

Cited by 18 publications

(41 citation statements)

References 27 publications

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“…structural states) within a protein complex as well as the population of probes in each structural state (29,30). With A N -CaM, the TR-FRET data corresponding to both RyR isoforms (Fig.…”

Section: Effect Of S100a1 and Cam On [mentioning

confidence: 99%

S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex

Rebbeck

Nitu

Rohde

et al. 2016

Journal of Biological Chemistry

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S100A1 has been suggested as a therapeutic agent to enhance myocyte Ca 2؉ cycling in heart failure, but its molecular mode of action is poorly understood. Using FRET, we tested the hypothesis that S100A1 directly competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. . We conclude that CaM and S100A1 can concurrently bind to and functionally modulate RyR1 and RyR2, but this does not involve direct competition at the RyR CaM binding site.

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“…structural states) within a protein complex as well as the population of probes in each structural state (29,30). With A N -CaM, the TR-FRET data corresponding to both RyR isoforms (Fig.…”

Section: Effect Of S100a1 and Cam On [mentioning

confidence: 99%

S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex

Rebbeck

Nitu

Rohde

et al. 2016

Journal of Biological Chemistry

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“…Previously, we used a different two-color pump, the P-type ATPase SERCA, to detect activator/inhibitor candidates in a high-throughput screen of a library of drug-like compounds (Gruber et al, 2014). The present data suggest that this approach can be successfully generalized to ABC proteins and to other families of transporters.…”

Section: Potential Application For Drug Discoverymentioning

confidence: 69%

“…The optimal cell line was .98% positive for GFP fluorescence measured by flow cytometry. Screening of the NIH Clinical Collection (NCC; a library of 446 drug-like compounds) was performed as previously described (Gruber et al, 2014). The NCC is a library of unique compounds that is ideal for small-scale trial screens.…”

Section: Methodsmentioning

confidence: 99%

“…To evaluate whether two-color MRP1 could be useful for identification of novel substrates or inhibitors, we performed a FRET-based screen of the NCC library of compounds that are known to be biologically active. We applied test compounds to live cells in a 384-well assay format as previously described (Gruber et al, 2014) and selected hits that altered the fluorescence lifetime of the GFP donor on MRP1 in repeated screens of the library. Two independent screens identified 20 and 34 hits, respectively, with 13 hits in common between the two screens.…”

Section: Potential Application For Drug Discoverymentioning

confidence: 99%

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ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

et al. 2015

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Multidrug resistance protein 1 (MRP1) actively transports a wide variety of drugs out of cells. To quantify MRP1 structural dynamics, we engineered a "two-color MRP1" construct by fusing green fluorescent protein (GFP) and TagRFP to MRP1 nucleotide-binding domains NBD1 and NBD2, respectively. The recombinant MRP1 protein expressed and trafficked normally to the plasma membrane. Two-color MRP1 transport activity was normal, as shown by vesicular transport of induces a large-amplitude conformational change that brings the NBDs into closer proximity. FRET was further increased by substrate in the presence of ATP but not by substrate alone. The data suggest that substrate binding is required to achieve a fully closed and compact structure. ATP analogs bind MRP1 with reduced apparent affinity, inducing a partially closed conformation. The results demonstrate the utility of the two-color MRP1 construct for investigating ATP-binding cassette transporter structural dynamics, and it holds great promise for high-throughput screening of chemical libraries for unknown activators, inhibitors, or transportable substrates of MRP1.

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“…Advances with cell sorting, single cell RNA sequencing, and computational methods revealed a rich diversity of cell identities and the existence of previously unknown cell subtypes (2,3). Single molecule tracking, correlation spectroscopy, and time-resolved fluorescence energy transfer protein dynamics and interactions in cells in unprecedented detail and allow high-throughput screening for new protein modulators (4,5). This thematic series aims to add to the excitement by highlighting recent methodological advances in three areas that differ markedly with respect to their scale and specific experimental goals but that, together, bring us closer to the ultimate goal of learning about cell function at the atomic level.…”

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confidence: 99%

Introduction to the Minireview Series on Modern Technologies for In-cell Biochemistry

Lutsenko

2016

Journal of Biological Chemistry

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The last decade has seen enormous progress in the exploration and understanding of the behavior of molecules in their natural cellular environments at increasingly high spatial and temporal resolution. Advances in microscopy and the development of new fluorescent reagents as well as genetic editing techniques have enabled quantitative analysis of protein interactions, intracellular trafficking, metabolic changes, and signaling. Modern biochemistry now faces new and exciting challenges. Can traditionally "in vitro" experiments, e.g. analysis of protein folding and conformational transitions, be done in cells? Can the structure and behavior of endogenous and/or nontagged recombinant proteins be analyzed and altered within the cell or in cellular compartments? How can molecules and their actions be studied mechanistically in tissues and organs? Is personalized cellular biochemistry a reality? This thematic series summarizes recent studies that illustrate some first steps toward successfully answering these modern biochemical questions. The first minireview focuses on utilization of three-dimensional primary enteroids and organoids for mechanistic studies of intestinal biology with molecular resolution. The second minireview describes application of single chain antibodies (nanobodies) for monitoring and regulating protein dynamics in vitro and in cells. The third minireview highlights advances in using NMR spectroscopy for analysis of protein folding and assembly in cells.The ultimate goal of biochemistry is to understand how molecules work in a complex cellular environment at atomic level. Although we are still far away from this goal, great progress has been made toward characterization of metabolic identities of various cells and the behavior of many essential molecules in diverse intracellular contexts. Chemically induced dimerization of recombinant proteins has enabled dissection of signaling events in distinct intracellular locations with fine temporal resolution (1). Advances with cell sorting, single cell RNA sequencing, and computational methods revealed a rich diversity of cell identities and the existence of previously unknown cell subtypes (2, 3). Single molecule tracking, correlation spectroscopy, and time-resolved fluorescence energy transfer protein dynamics and interactions in cells in unprecedented detail and allow high-throughput screening for new protein modulators (4, 5). This thematic series aims to add to the excitement by highlighting recent methodological advances in three areas that differ markedly with respect to their scale and specific experimental goals but that, together, bring us closer to the ultimate goal of learning about cell function at the atomic level.In the first minireview of this thematic series (6), Zachos et al. (6) focus on the advantages of primary human enteroids and organoids for studies of intestinal physiology and pathobiology. In recent years, it has become increasingly clear that studies directed toward the understanding and treatment of human disorders require exp...

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Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

Cited by 18 publications

References 27 publications

S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex

S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex

ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

Introduction to the Minireview Series on Modern Technologies for In-cell Biochemistry

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