Quantifying the Efficiency of Various FRET Constructs Using Optimis™

Stoneman, Michael R.; Patowary, Suparna; Singh, Deo R.; Komarova, L V; Westrick, L G; Oliver, Julie A.; Raicu, Valerică

doi:10.2144/000113828

Cited by 3 publications

(3 citation statements)

References 9 publications

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“…These also confirmed that the two forms of M 3 R were in close apposition, and the specificity of the PLA was In light of all these findings, and with the application of Occam's razor, we hypothesize that the rhombus tetramer is the largest quaternary structure formed by M 3 Rs at the plasma membrane. Separate control experiments, which incorporated FRET standards, confirmed that the detected oligomers are not artefacts potentially introduced by the methods of the present study [15,34,35]. 'Experimental' bars represent the areas under each Gaussian peak in the individual E app histograms, as in Figure 1(C), averaged over several cells.…”

Section: Determination Of the Largest Quaternary Structure Of M 3 R Asupporting

confidence: 70%

The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane

Patowary

Álvarez-Curto

et al. 2013

Biochemical Journal

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106

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The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M(3)Rs (muscarinic M(3) acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M(3)R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

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Section: Determination Of the Largest Quaternary Structure Of M 3 R Asupporting

confidence: 70%

The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane

Patowary

Álvarez-Curto

et al. 2013

Biochemical Journal

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106

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“…Each fusion protein was expressed in Chinese hamster ovary (CHO) cells, and the FRET efficiencies were measured at the level of single pixels using an imaging system designed for optical microspectroscopy (23,30). According to the theory (22), the apparent FRET efficiency of a fluorescent quadruplex containing multiple donors and acceptors (e.g., ADAA) can be predicted from measurements on analogs that contain a single donor and acceptor (e.g., NDAN, NDNA, and ADNN); similarly, the apparent FRET efficiency of a fluorescent triplex such as ADA can be predicted from the pairwise FRET efficiency measured for the duplexes DA and AD.…”

Section: Introductionmentioning

confidence: 99%

Experimental Verification of the Kinetic Theory of FRET Using Optical Microspectroscopy and Obligate Oligomers

Patowary

Pisterzi

Biener

et al. 2015

Biophysical Journal

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Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

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“…Chen and co-workers measured the energetics of the GpA transmembrane domain dimerization in vesicles derived from mammalian membrane (11). Stoneman et al used commercially available Premo Cameleon FRET biosensors (Life Technologies, Carlsbad, CA) to probe induced calcium-ion concentration changes within the cell (12).…”

Section: Introductionmentioning

confidence: 99%

FRET Spectrometry: A New Tool for the Determination of Protein Quaternary Structure in Living Cells

Raicu

Singh

2013

Biophysical Journal

100

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Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, E(app), across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the E(app) distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method--akin to that of mass spectrometry--for sorting protein complexes according to their size and shape.

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Quantifying the Efficiency of Various FRET Constructs Using Optimis™

Cited by 3 publications

References 9 publications

The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane

The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane

Experimental Verification of the Kinetic Theory of FRET Using Optical Microspectroscopy and Obligate Oligomers

FRET Spectrometry: A New Tool for the Determination of Protein Quaternary Structure in Living Cells

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