2016
DOI: 10.1039/c5ib00202h
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Fully quantified spectral imaging revealsin vivomembrane protein interactions

Abstract: Here we introduce the Fully Quantified Spectral Imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. W… Show more

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Cited by 90 publications
(304 citation statements)
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References 56 publications
(127 reference statements)
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“…Biophysical information about drug targets can yield better mechanistic understanding of their function and ultimately can allow for more accurate prediction of drug action and function. Recently, advances in microscopy have complemented the biochemical assays traditionally used in membrane protein research, producing new insights about membrane protein function (6,7,11,23,24,43,(55)(56)(57). In this paper, we build on our previous work to develop and implement a quantitative FRET assay for examining RTK heterodimers.…”
Section: Discussionmentioning
confidence: 99%
“…Biophysical information about drug targets can yield better mechanistic understanding of their function and ultimately can allow for more accurate prediction of drug action and function. Recently, advances in microscopy have complemented the biochemical assays traditionally used in membrane protein research, producing new insights about membrane protein function (6,7,11,23,24,43,(55)(56)(57). In this paper, we build on our previous work to develop and implement a quantitative FRET assay for examining RTK heterodimers.…”
Section: Discussionmentioning
confidence: 99%
“…Protoplasts transfected by single plasmids were used as controls. Acquisition of spectrally resolved fluorescence images, spectral unmixing of donor, acceptor, and autofluorescence signals, as well as determination of FRET efficiency were performed as described previously (Raicu and Singh, 2013;King et al, 2016). Briefly, cells coexpressing EMS1-CFP and SERK1-YFP (controls: cells expressing only EMS1-CFP or SERK1-YFP) were imaged using an optical microspectroscope (OptiMiS) with the line-scan excitation powered by a femtosecond laser emitting near-infrared light pulses with tunable emission wavelength between 780 and 1,040 nm (Raicu et al, 2009;Biener et al, 2013).…”
Section: Fret Assaymentioning
confidence: 99%
“…Such specific interactions between the two D7 domains could stabilize VEGFR2 unliganded dimers. In our previous work, we therefore asked if the D731-R726 salt bridge plays a role in dimer stabilization, in the absence of ligand (18). We reasoned that if this salt bridge forms in the absence of ligand and if it stabilizes the dimer, its elimination will reduce, or even eliminate, VEGFR2 dimerization.…”
Section: Introductionmentioning
confidence: 99%
“…If, however, the salt bridge does not form and plays no role in unliganded dimerization, its elimination will have no effect on VEGFR2 dimerization. To investigate which of these possibilities occur, we introduced a D731A mutation in a VEGFR2 construct in which the intracellular domain was substituted with a fluorescent protein to allow for FRET detection, and we assessed the effect of the D731A mutation on its dimerization in the absence of ligand (18). To our surprise, none of the two hypothesized scenarios occurred in these experiments.…”
Section: Introductionmentioning
confidence: 99%
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