Simple Estimation of Förster Resonance Energy Transfer (FRET) Orientation Factor Distribution in Membranes

Loura, Luís M. S.

doi:10.3390/ijms131115252

Cited by 75 publications

(67 citation statements)

References 53 publications

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“…S8). Consistent with existing analytical treatments and simulations (38,39), constrained geometry did not substantially change the predicted quenching curves. The major effect was an effective reduction of R 0 , bringing the region of positive slope of ΔF into a position (∼5 Å from the membrane) that cannot be reached due to the size of the donor.…”

Section: Voltage-dependent Quenching Of Yfp In the Intracellular Regisupporting

confidence: 70%

Structural rearrangement of the intracellular domains during AMPA receptor activation

Zachariassen

Katchan

Jensen

et al. 2016

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

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α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/ YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double-and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.T he α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (iGluRs) reside in postsynaptic membranes in the central nervous system (1). Like the other major iGluR subtypes, the N-methyl-Daspartate receptors (NMDARs) and kainate receptors, AMPARs are ligand-gated cation channels formed by hetero-or homotetrameric assembly of the GluA1 to GluA4 subunits into a membranespanning receptor with an integral ion channel. The energy from binding of the excitatory neurotransmitter glutamate at extracellular sites triggers AMPARs to passage through functional states that can include ion channel activation, deactivation, and desensitization. The timing and interplay between the necessarily distinct conformations of these states determine both basal transmission and short-term plasticity of the postsynapse (2-4).Structures of AMPARs captured in various functional states using X-ray crystallography and cryoelectron microscopy have recently become available (5-9). Overall, the structures confirm three distinct structural layers (Fig. 1). The amino-terminal domain (ATD) and the ligand-binding domain (LBD), both distal to the membrane, and the transmembrane ion channel are each formed from four copies of the respective domain from each subunit. However, a fourth region of the receptor extends from the intracellular side of the membrane. This part of the receptor includes intracellular loops between the transmembrane helices and the variable C-terminal domain (CTD), and remains entirely unresolved in the presently available AMPAR structures.The GluA2 structures and previous results from biochemical, biophysical, and crystallographic studies of isolated subunits and fu...

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Section: Voltage-dependent Quenching Of Yfp In the Intracellular Regisupporting

confidence: 70%

Structural rearrangement of the intracellular domains during AMPA receptor activation

Zachariassen

Katchan

Jensen

et al. 2016

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

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“…The overlap between the emission spectrum of ANAP and the absorption spectrum of Co 2+ -NTA, together with the quantum yield of ANAP (0.22; Fig. S3), predicts an R 0 of 12.8 Å for mobile randomly oriented donors and acceptors ( 2 = 2/3; Fung and Stryer, 1978;Loura, 2012).…”

Section: Expression Of L-anap-incorporated Trpv1 Constructs As Functimentioning

confidence: 99%

“…8 A), respectively, in stabilization buffer, our estimate of 0.22 for the quantum yield of ANAP in stabilization buffer (Fig. S3), and assuming  2 of 2/3 (Fung and Stryer, 1978;Loura, 2012). The density of C18-NTA-Co 2+ incorporated into the plasma membrane was assumed to be 0.002 molecules/ Å 2 from our previous experiments with rhodamine B and C18-NTA-Co 2 (Gordon et al, 2016).…”

Section: +mentioning

confidence: 99%

Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET

Zagotta¹,

Gordon²,

Senning³

et al. 2016

J Cell Biol

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Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co 2+ bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

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“…4 A for Co 2+ alone (gray trace) and for Co 2+ chelated with nitrilotriacetic acid (Co 2+ -NTA; black trace). The overlap between the emission spectrum of rhodamine B (red trace) and the absorption spectrum of Co 2+ -NTA together with the quantum yield of rhodamine B (0.3; Magde et al, 1999), predicts an R 0 of 12.0 Å for mobile randomly oriented donors and acceptors ( 2 = 2/3; Fung and Stryer, 1978;Loura, 2012). …”

Section: Online Supplemental Materialsmentioning

confidence: 99%

“…R 0 for rhodamine B and Co 2+ -NTA was calculated as 12.0 Å based on our own emission and absorption spectra of rhodamine B and Co 2+ -NTA, respectively, in stabilization buffer (see Fig. 4 A) and assuming  2 of 2/3 (Fung and Stryer, 1978;Loura, 2012). A fluorescence lifetime,  D , of 1.5 ns was used for rhodamine B (see Fig.…”

Section: Epifluorescent Patch Imagingmentioning

confidence: 99%

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

Gordon¹,

Senning²,

Aman³

et al. 2016

J Cell Biol

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The plasma membrane is a major site of signal detection, transduction, and propagation in cells. Understanding cell signaling dynamics at the plasma membrane requires approaches that can detect changes in membrane architecture over molecular distances (10-100 Å) and biological time scales (milliseconds to seconds). Methods that provide access to dynamics at the intracellular surface of the plasma membrane would be especially powerful. Fluorescence resonance energy transfer (FRET) was first applied to studies of membrane dynamics by Keller et al. (1977), who recognized that fluorescent probes incorporated into membranes could be used as an indicator of mixing of the two membranes during membrane fusion. FRET occurs when the emission spectrum of a donor fluorophore overlaps with the absorption spectrum of an acceptor, and the donor and acceptor are in close proximity (Lakowicz, 2006;Taraska and Zagotta, 2010). FRET is steeply distance dependent, and each donor-acceptor pair has a characteristic distance at which the FRET efficiency is 50% (R 0 ), where the amount of FRET is optimally sensitive to changes in distance. The R 0 value for fluorescein and rhodamine, for example, is 60 Å (Delgadillo and Parkhurst, 2010), making them an ideal FRET pair for quantifying membrane fusion (Keller et al., 1977).Transition metal ion FRET (tmFRET) is a method to probe the shorter distances typical of the conformational landscape of proteins (Taraska and Zagotta, 2010). tmFRET is a form of FRET that uses a fluorophore as a Correspondence to Sharona E. Gordon: s e g @ u w . e d u ; or William N. Zagotta: z a g o t t a @ u w . e d u Abbreviations used in this paper: FRET, fluorescence resonance energy transfer; PCF, patch-clamp fluorometry; TIRF, total internal reflection fluorescence; tmFRET, transition metal ion FRET.donor and a nonfluorescent transition metal ion as an acceptor (Latt et al., 1970(Latt et al., , 1972Horrocks et al., 1975;Richmond et al., 2000;Sandtner et al., 2007; Taraska et al., 2009a,b;Yu et al., 2013). Transition metal ions such as Ni 2+ , Co 2+ , and Cu 2+ have broad absorption spectra that overlap the emission spectra of visible light fluorophores (see Miessler and Tarr [1999] pages 361-380 for a discussion of electronic spectra of coordination compounds). Transition metals can therefore accept energy transfer from a nearby donor fluorophore and quench the donor's fluorescence. The degree of quenching is a direct measure of the FRET efficiency (Lakowicz, 2006;Taraska et al., 2009a). The FRET efficiency is steeply dependent on distance between the donor and acceptor, allowing tmFRET to serve as a molecular ruler for distances in the membrane. The main advantages of tmFRET over classical FRET methods are (a) R 0 is very short (10-20 Å), allowing short-range interactions to be studied; (b) the transition metals have multiple transition dipoles, reducing the orientation dependence of FRET (Horrocks et al., 1975); (c) the metals can be reversibly bound to native and introduced binding sites; and (d) different...

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Simple Estimation of Förster Resonance Energy Transfer (FRET) Orientation Factor Distribution in Membranes

Cited by 75 publications

References 53 publications

Structural rearrangement of the intracellular domains during AMPA receptor activation

Structural rearrangement of the intracellular domains during AMPA receptor activation

Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

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