Calculation on fluorescence resonance energy transfer on surfaces

Dewey, T. Gregory; Hammes, Gordon G.

doi:10.1016/s0006-3495(80)85033-8

Cited by 188 publications

(124 citation statements)

References 14 publications

(19 reference statements)

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“…FRET measurements for a third GPI-anchored protein, 5Ј NT, the protein we studied previously in MDCK cells (Kenworthy and Edidin, 1998), yielded slightly higher values of E than did measurements for CD59 in Fao cells ( Figure 5B). These values are similar to, but slightly higher than, the predicted E of ϳ1% we calculated from theoretical equations (Wolber and Hudson, 1979;Dewey and Hammes, 1980) assuming r ϭ 0 and a surface density of ϳ100 monomers of 5Ј NT/ m 2 at the Fao cell plasma membrane (Howell et al, 1987). 5Ј NT exhibited a relatively weak cell-to-cell dependence of E on surface density, but similar curves were obtained for D:A of 1:1-1:3 (our unpublished results).…”

Section: Fret Microscopy Of Lipid Raft Componentssupporting

confidence: 85%

“…For example, CTXB gave systematically higher E values than did antibody-labeled GPI-anchored proteins when compared at the same acceptor fluorescence intensity ( Figure 5B). Such behavior is predicted for randomly distributed molecules of different sizes or geometries (Wolber and Hudson, 1979;Dewey and Hammes, 1980). These findings provide evidence that the FRET results are specific and that we should be able to detect clustered membrane proteins.…”

Section: Discussionsupporting

confidence: 63%

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High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Kenworthy

Petranova

Edidin

2000

MBoC

406

311

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“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

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Section: Fret Microscopy Of Lipid Raft Componentssupporting

confidence: 85%

Section: Discussionsupporting

confidence: 63%

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Kenworthy

Petranova

Edidin

2000

MBoC

406

311

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“…Our data could not be fit by theoretical curves (23,24). This is because the dependence of FRET on YFP surface density is not linear, and because a number of the variables, needed for a fit, are unknown.…”

Section: Fluorescence Microscopy and Fret Measurementsmentioning

confidence: 97%

Clustering of Peptide-Loaded MHC Class I Molecules for Endoplasmic Reticulum Export Imaged by Fluorescence Resonance Energy Transfer

Pentcheva

Edidin

2001

The Journal of Immunology

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Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.

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“…This FRET metric offers three distinct advantages that appeal to the experimentalist: (i) Only a single measurement is needed (i.e., it is not necessary to prepare two parallel samples labeled with either donor alone or donor + acceptor); (ii) Dex A F measurements tend to be more robust, 5 In order to provide for the interpretation of experimental results, freely-diffusing probe studies must resort to a theoretical framework in order to relate variations in the FRET metric to variations in probe distributions. Whereas a common ( ) D E χ framework has long been in use for membrane studies, 6,7,8 F have therefore employed phenomenological models or else resorted to computer simulation. 5 In the experiments presented here, we have explored the utility of simple Stern-Volmer (S-V) probe-dependence expressions-for both…”

Section: Introductionmentioning

confidence: 99%

Stern-Volmer modeling of steady-state Förster energy transfer between dilute, freely diffusing membrane-bound fluorophores

Buboltz

Bwalya

Reyes

et al. 2007

The Journal of Chemical Physics

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INTRODUCTIONThe phenomenon of Förster resonance energy transfer (FRET) between two fluorescent chromophores is widely employed for a variety of purposes. 1,2 In this form, FRET may perhaps be best known as a "spectroscopic ruler," serving in applications that exploit the famous inverse sixthorder distance dependence 3 of the so-called transfer efficiency, E . This particular FRET metric is the fractional decrease in donor fluorescence due to acceptor quenching, aso the determination of E requires two separate measurements: donor fluorescence in the presence and absence of acceptor. During the last decade, FRET has seen increasing application in studies of biological membranes, both in model systems 4 and living cells. 2 In these studies, membranes are labeled with two populations of membrane-associated fluorophores (i.e., donor and acceptor probes), and the observed FRET signal is interpreted in terms of either membrane phase behavior or specific interactions between membrane components.Although most of these biomembrane FRET studies have been based on measurements of E , others have chosen to use an alternative metric: donor-excited acceptor fluorescence, of E ; and (iii) whereas measurements of E are sensitive to variations in acceptor concentration only:measurements are sensitive to variations in both probe concentrations:In order to provide for the interpretation of experimental results, freely-diffusing probe studies must resort to a theoretical framework in order to relate variations in the FRET metric to variations in probe distributions. Whereas a common Eχ -using six different combinations of FRET probes and membrane environments. Of course, analyses based on the S-V model are normally applied only to experiments involving collisional quenching. But dilute acceptor concentration is one condition under which Forster kinetics are known to approach the Stern-Volmer limit, b9 so under these circumstances it is also reasonable to employ an S-V model to describe FRET.Our original goal was simply to evaluate the useful limits of an S-V expression forso that we could use it in our FRET-based studies of membrane phase behavior. 10 However, over the course of our research we have discovered that S-V expressions can safely be used to describe both FRET metrics within acceptor-concentration ranges that are conveniently defined by an easily measured parameter: the Stern-Volmer quenching constant. Moreover, we have seen that S-V predictions can even work well up to remarkably b The other condition being "statistical mixing" of excited-state donors and acceptors due to rapid diffusion or excitation migration.[ F has been gaining in popularity for practical reasons among experimentalists who study biomembranes. Here, for the special case of membrane-bound fluorophores, we present a substantial body of experimental evidence that justifies the use of simple Stern-Volmer expressions when modeling either FRET metric under diluteprobe conditions. We have also discovered a dilute-regime correspondence between our Stern-Vol...

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Calculation on fluorescence resonance energy transfer on surfaces

Cited by 188 publications

References 14 publications

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Clustering of Peptide-Loaded MHC Class I Molecules for Endoplasmic Reticulum Export Imaged by Fluorescence Resonance Energy Transfer

Stern-Volmer modeling of steady-state Förster energy transfer between dilute, freely diffusing membrane-bound fluorophores

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