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

Abstract

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...

A consistent picture of TRPV1 activation emerges from molecular simulations and experiments

“…This limitation is particularly severe in the case of TRPV1 whose activity shows exquisite dependency on numerous environmental factors, such as lipid composition (8,(63)(64)(65)(66)(67), extracellular concentration of Na + (11) and the presence of cholesterol or other fatty acids (68)(69)(70)(71). Accordingly, advanced experimental techniques, such as cell unroofing (72,73) and incorporation of novel unnatural amino acids (42,73), are being constantly developed to overcome these limitations and introduce only minimal perturbations to the native environment of the channel. Despite the great progress, these techniques have focused so far only on specific aspects of TRPV1 activation.…”

Visualizing conformational dynamics of proteins in solution and at the cell membrane

“…Cells were imaged and unroofed as previously described (17,20). Briefly, coverslips were mounted in a homemade chamber on a microscope stage and perfused for several minutes with HBR via gravity-flow perfusion.…”

“…For L-Anap excitation we used a 375/28 nm excitation filter and a 480/50 nm emission filter. Each experiment commenced with a pre-bleaching step, comprised of a seven-second exposure of the coverslip to L-Anap excitation light (20). Images for analysis were collected using exposures ranging from 100 ms to 500 ms, depending on sample intensity, using a QuantEM EMCCD camera (Photometrics, Tuscon, AZ) with readout speed of 5 MHz and the multiplier set to 20.…”

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The emerging role of physical modeling in the future of structure determination

“…Transition metal ion FRET (tmFRET) measures the distance between small-molecule fluorophores and a nonfluorescent transition metal. Because it provides short range distances, and because different metals have different absorptions, the method is tunable for a range of distances (10-20Å) [78] and has been used to study membrane proteins [79].…”