Steady-state probe-partitioning fluorescence resonance energy transfer: A simple and robust tool for the study of membrane phase behavior

Buboltz, Jeffrey T.

doi:10.1103/physreve.76.021903

Cited by 31 publications

(42 citation statements)

References 29 publications

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“…The theoretical framework of the steady-state probe-partitioning FRET in a 2D surface is well described (Buboltz, 2007). The trend in inducing PIP 2 cluster formation in a bilayer membrane and a low ionic strength buffer follows the order: Ca 2+ ≫ Mg 2+ > Zn 2+ .…”

Section: Experimental Studies Of Cation-mediated Interactions Amonmentioning

confidence: 99%

Counterion-mediated cluster formation by polyphosphoinositides

Wang

Slochower

Janmey

2014

Chemistry and Physics of Lipids

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Polyphosphoinositides (PPI) and in particular PI(4,5)P2, are among the most highly charged molecules in cell membranes, are important in many cellular signaling pathways, and are frequently targeted by peripheral polybasic proteins for anchoring through electrostatic interactions. Such interactions between PIP2 and proteins containing polybasic stretches depend on the physical state and the lateral distribution of PIP2 within the inner leaflet of the cell's lipid bilayer. The physical chemical properties of PIP2 such as pH-dependent changes in headgroup ionization and area per molecule as determined by experiments together with molecular simulations that predict headgroup conformations at various ionization states have revealed the electrostatic properties and phase behavior of PIP2-containing membranes. This review focuses on recent experimental and computational developments in defining the physical chemistry of PIP2 and its interactions with counterions. Ca2+-induced changes in PIP2 charge, conformation, and lateral structure within the membrane are documented by numerous experimental and computational studies. A simplified electrostatic model successfully predicts the Ca2+-driven formation of PIP2 clusters but cannot account for the different effects of Ca2+ and Mg2+ on PIP2-containing membranes. A more recent computational study is able to see the difference between Ca2+ and Mg2+ binding to PIP2 in the absence of a membrane and without cluster formation. Spectroscopic studies suggest that divalent cation- and multivalent polyamine-induced changes in the PIP2 lateral distribution in model membrane are also different, and not simply related to the net charge of the counterion. Among these differences is the capacity of Ca2+ but not other polycations to induce nm scale clusters of PIP2 in fluid membranes. Recent super resolution optical studies show that PIP2 forms nanoclusters in the inner leaflet of a plasma membrane with a similar size distribution as those induced by Ca2+ in model membranes. The mechanisms by which PIP2 forms nanoclusters and other structures inside a cell remain to be determined, but the unique electrostatic properties of PIP2 and its interactions with multivalent counterions might have particular physiological relevance.

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Section: Experimental Studies Of Cation-mediated Interactions Amonmentioning

confidence: 99%

Counterion-mediated cluster formation by polyphosphoinositides

Wang

Slochower

Janmey

2014

Chemistry and Physics of Lipids

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“…DHE and Fast DiO were chosen as a FRET pair, at 1/100 and 1/700 fractions in total lipid. Light scattering and fluorescence background were controlled as described previously (71,72). Lipids were mixed in chloroform to 120 nmol, and the organic solvent was replaced by buffer using rapid solvent exchange (RSE) (68) and then sealed GUV samples described here were prepared by gentle hydration (38,73).…”

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confidence: 99%

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Wen

Dick

Feigenson

et al. 2016

J Virol

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The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCERetroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.T he retroviral structural protein Gag provides the primary driving force for virus assembly at the inner leaflet of the plasma membrane (PM), and Gag alone is sufficient to assemble into virus-like particles (VLPs) in cells (1) and also in vitro (2). Gag is synthesized in the cytosol and then is targeted to the PM by the N-terminal domain, MA. How Gag interacts with the inner leaflet of the PM and how the biophysical properties of the PM...

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“…The measured fluorescence intensities from the fluorophores were significantly lower in the absence of micelles (Figure S5), consistent with prior literature showing that the partitioning of fluorophores increases the fluorescence intensity, due to a higher local concentration. 49 …”

Section: Resultsmentioning

confidence: 99%

Fluorophore exchange kinetics in block copolymer micelles with varying solvent–fluorophore and solvent–polymer interactions

et al. 2016

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Fluorescence spectroscopy was employed to characterize the kinetics of guest exchange in diblock copolymer micelles composed of poly(ethylene oxide-b-ε-caprolactone) (PEO-PCL) diblock copolymers in water/tetrahydrofuran (THF) mixtures which encapsulated fluorophores. The solvent composition (THF content) of the micelle solution was varied as a means of modulating the strength of interactions between the fluorophore and solvent as well as between the micelle core and solvent. A donor-acceptor fluorophore pair was employed consisting of 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO, the donor) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, the acceptor). Through the process of Förster resonance energy transfer (FRET), energy was transferred from the donor to acceptor when the fluorophores were in close proximity. A micelle solution containing DiO was mixed with a micelle solution containing DiI at t=0, and the emission spectra of the mixed solution were monitored over time (at an excitation wavelength optimized for the donor). In micelle solutions containing 5 and 10 vol% THF in the bulk solvent, an increase in the acceptor peak intensity maximum occurred over time in the post-mixed solution, accompanied by a decrease in the donor peak intensity maximum, indicating the presence of energy transfer from the donor to the acceptor. At long times, the FRET ratios (acceptor peak intensity divided by the sum of the acceptor and donor peak intensities) were indistinguishable from that determined from pre-mixed micelle solutions of the same THF content (in pre-mixed solutions, DiO and DiI were encapsulated within the same micelle cores). In the micelle solution containing 20 vol% THF, the fluorophore exchange process occurred too quickly to be observed (the FRET ratios measured from the solutions mixed at t=0 were commensurate to that measured from the pre-mixed solution). A time constant describing the guest exchange process was extracted from the time-dependence of the FRET ratio through fit of an exponential decay. An increase in the THF content in the micelle solution resulted in a decrease in the time constant, and the time constant varied over five orders of magnitude as the THF content was varied from 5–20 vol%.

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Steady-state probe-partitioning fluorescence resonance energy transfer: A simple and robust tool for the study of membrane phase behavior

Cited by 31 publications

References 29 publications

Counterion-mediated cluster formation by polyphosphoinositides

Counterion-mediated cluster formation by polyphosphoinositides

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Fluorophore exchange kinetics in block copolymer micelles with varying solvent–fluorophore and solvent–polymer interactions

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