The structure and function of fluorescent proteins

Sample, Vedangi; Newman, Robert H.; Zhang, Jin

doi:10.1039/b913033k

Cited by 114 publications

(103 citation statements)

References 49 publications

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“…FRET occurs between eCFP and mCitrine, variants of CFP and YFP, which are used because of their increased brightness and fluorophore stability (16). When CFP and YFP are in close proximity (≤8 nm) excitation of CFP results in significant FRET from CFP to YFP.…”

Section: Resultsmentioning

confidence: 99%

Systematic control of protein interaction using a modular ER/K α-helix linker

Sivaramakrishnan

Spudich

2011

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

124

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Cellular functions of proteins are strongly influenced by their interactions with other proteins. The frequency of protein interactions is a function of the local concentration of two proteins and their affinity for one another. When two proteins are tethered together, the link between them influences their effective concentrations and therefore the frequency of their interaction. Currently no methods exist to systematically vary the effective concentration within this intramolecular interaction. Here we outline a modular, genetically encoded linker, namely, an ER∕K [genetically encoded polypeptide motif based on alternating sequence of approximately four glutamic acid (E) followed by approximately four arginine (R) or lysine (K) residues] single α-helix that can be used to regulate the frequency of interaction between two proteins, or between a protein and a peptide, one at each end. We exploit the wide range of interaction affinities between calmodulin and its binding peptides, combined with FRET to determine the effect of the ER∕K α-helix in regulating protein interactions. We find that increasing the length of the ER∕K α-helix reduces the on rate of the intramolecular interaction without significantly affecting the off rate, regardless of the affinity of the bimolecular interaction. We outline a genetically encoded approach to determine the dissociation constant for both moderate (micromolar K d ) and strong (nanomolar K d ) protein interactions. Our studies demonstrate the use of the ER∕K α-helix to systematically engineer FRET biosensors that detect changes in concentration or affinity of interacting proteins, and modulate enzyme autoinhibition. Our findings are consistent with the ER∕K α-helix as a worm-like chain with rare, stochastic breaks in the helix backbone that may account for the behavior of myosin VI stepping along actin.systematic protein affinity strength | modulation | cell signaling | biochemistry I nteractions between proteins are essential for cellular function. The association of proteins facilitates their localization to, and therefore function in, specific subcellular compartments (1). Proteins often associate to form a macromolecular complex that can perform functions that the individual components cannot (2). Brief bimolecular interactions among a series of proteins are characteristic of signaling cascades that are essential to propagate and amplify an extracellular signal across the cell and elicit an appropriate response (3). Interactions between proteins are dictated by their binding affinity and the local cellular concentrations of the proteins involved. In addition to bimolecular interactions between proteins, many proteins have intramolecular interactions between distinct domains that regulate their function (4). Such intramolecular interactions within a protein in turn are regulated by the free solution binding affinity of the two domains and the effective concentration experienced by the two domains within the protein.Regulation of intermolecular and intramolecular protein int...

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Section: Resultsmentioning

confidence: 99%

Systematic control of protein interaction using a modular ER/K α-helix linker

Sivaramakrishnan

Spudich

2011

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

124

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“…1- 4 The optical properties of GFP can be attributed to a chromophore which is based on p-hydroxybenzylidene-2, dimethylimidazolinone (HBDI, Fig. 1).…”

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confidence: 99%

Photoelectron spectroscopy of the model GFP chromophore anion

Horke

Verlet

2012

Phys. Chem. Chem. Phys.

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“…Given their genetic encodability, along with the relative ease of recombinant DNA technology, FP tagging soon replaced the use of fluorescent analogs (Whitaker, 2000). Meanwhile, improved spectrally distinct FPs (Day and Davidson, 2009;Sample et al, 2009;Shaner et al, 2005) enabled the construction of FRET-based biosensors, with changes in FRET actively reporting on biochemical events in cells. The first such biosensor, the Ca 2+ probe Cameleon, featured CaM and the M13 peptide sandwiched between either GFP and BFP or CFP and YFP (Miyawaki et al, 1997).…”

Section: Genetically Encoded Biosensors To Monitor Signaling Dynamicsmentioning

confidence: 99%

Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

Sample

Mehta

Zhang

2014

Journal of Cell Science

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In this Commentary, we discuss two sets of genetically encoded molecular tools that have significantly enhanced our ability to observe and manipulate complex biochemical processes in their native context and that have been essential in deepening our molecular understanding of how intracellular signaling networks function. In particular, genetically encoded biosensors are widely used to directly visualize signaling events in living cells, and we highlight several examples of basic biosensor designs that have enabled researchers to capture the spatial and temporal dynamics of numerous signaling molecules, including second messengers and signaling enzymes, with remarkable detail. Similarly, we discuss a number of genetically encoded biochemical perturbation techniques that are being used to manipulate the activity of various signaling molecules with far greater spatial and temporal selectivity than can be achieved using standard pharmacological or genetic techniques, focusing specifically on examples of chemically driven and light-inducible perturbation strategies. We then describe recent efforts to combine these diverse and powerful molecular tools into a unified platform that can be used to elucidate the molecular details of biological processes that may potentially extend well beyond the realm of signal transduction.

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The structure and function of fluorescent proteins

Cited by 114 publications

References 49 publications

Systematic control of protein interaction using a modular ER/K α-helix linker

Systematic control of protein interaction using a modular ER/K α-helix linker

Photoelectron spectroscopy of the model GFP chromophore anion

Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

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