Fluorescence resonance energy transfer analysis of lipopolysaccharide in detergent micelles

Wiström, Christina Aurell; Jones, Gregory M.; Tobias, Peter S.; Sklar, Larry A.

doi:10.1016/s0006-3495(96)79642-x

Cited by 12 publications

(15 citation statements)

References 56 publications

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“…LPS of so-called rough mutant strains do not have the O-antigen, but, rather, they have only 2-15 nonrepeating ''core'' oligosaccharides (39), which are characterized by ''chemotypes'' with decreasing lengths of the ''core sugars'', known as Ra, Rb, Rc, Rd, and Re (40). R595, which is an Re LPS with two core sugars, has been reported to be chemically homogeneous from deoxycholate and sodium dodecyl sulfate gel electrophoresis studies (41)(42)(43), probably because of the lack of heterogeneous O-antigen. Taking advantage of this homogeneity and two primary amine groups in the molecule, R595 was chosen for derivatization with NBD.…”

Section: Rationale For Derivatization Of S Minnesota R595mentioning

confidence: 99%

A Novel Fluorescent Probe That Senses the Physical State of Lipid Bilayers

Sasaki

White

2009

Biophysical Journal

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Cell membrane lipids and proteins are heterogeneously distributed in the membrane plane. In recent years, much attention has been paid to the heterogeneous distribution of the lipid components, particularly the formation of cholesterol-rich domains that are thought to be important in signaling processes. This has led to renewed interest in the phase diagrams of complex lipid mixtures, such as three-component mixtures containing phospholipids and cholesterol. We report here a novel fluorescent probe (NBD-R595) that is useful for exploring the phase behaviors of one-, two-, and three-component large unilamellar vesicles. In one-component fluid-phase membranes, the probe has the expected spectral characteristic of monomeric 7-nitrobenzo-2-oxa-1,3-diazol, with a fluorescence maximum of 540 nm when excited at 470 nm. But below the gel-to-liquid crystalline phase transition temperature, an additional emission peak appears at approximately 610 nm, because of Förster resonance energy transfer from NBD-R595 monomers to NBD-R595 Jelley aggregates of limited size formed by the association of 7-nitrobenzo-2-oxa-1,3-diazol moieties. This may be the first report of Förster resonance energy transfer from a single fluorophore in two different physical states. In a test of the probe, we found NBD-R595 to be remarkably sensitive to the molar composition of large unilamellar vesicles formed from cholesterol, distearoylphosphatidylcholine, and dioleoylphosphatidylcholine.

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Section: Rationale For Derivatization Of S Minnesota R595mentioning

confidence: 99%

A Novel Fluorescent Probe That Senses the Physical State of Lipid Bilayers

Sasaki

White

2009

Biophysical Journal

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“…Excitation at 470 nm was used to minimize direct excitation of Fast-DiI (absorbance maximum, 549 nm). FITC-LPS concentrations were 30 nM in FITC and SBP was 100 M. Using an aggregation number of 155, and a critical micelle concentration (CMC) of 15 M with 85% of the detergent in micelles, gives a micelle concentration of 6 ϫ 10 Ϫ7 M (Herrmann, 1966;Aurell Wiström et al, 1996). The buffer consisted of 0.02 M Trizma, 0.15 M NaCl, pH 7.5.…”

Section: Steady-state Fluorescence Measurements In Micellesmentioning

confidence: 99%

“…where Q 0 is the quantum yield of the donor in the absence of acceptors, n is the index of refraction of the medium separating the donor and acceptor, and 2 is the dipole-dipole orientation factor. The method of Dale et al (1979), based on fluorescence polarization anisotropy measurements, was used to restrict the range of 2 (Aurell Wiström et al, 1996). The spectral overlap integral (J) was calculated for donor and acceptor pairs according to Aurell Wiström et al (1996).…”

Section: Calculating the Steady-state Fluorescence As A Function Of The Donor-to-micelle Surface Distancementioning

confidence: 99%

“…The method of Dale et al (1979), based on fluorescence polarization anisotropy measurements, was used to restrict the range of 2 (Aurell Wiström et al, 1996). The spectral overlap integral (J) was calculated for donor and acceptor pairs according to Aurell Wiström et al (1996). As the transfer efficiency is sensitive to the separation of donor and acceptor where the distances are contained in the interval 0 Յ R Յ 2R 0 , and the micelle radius is ϳ0.3R 0 , the micelle geometry needs to be taken into account (Ediger et al, 1984;Kaschke et al, 1988).…”

Section: Calculating the Steady-state Fluorescence As A Function Of The Donor-to-micelle Surface Distancementioning

confidence: 99%

“…We have recently examined FRET between acceptors embedded in a micelle and donors associated with amphipathic molecules extending above the micelle surface (Aurell Wiström et al, 1996). Because of the general potential of such an approach for revealing details about molecular conformation, we have analyzed the system in more detail.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Vertical Fluorescence Resonance Energy Transfer from the Surface of a Small-Diameter Sphere

Jones

Wofsy

Aurell

et al. 1999

Biophysical Journal

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Fluorescence resonance energy transfer (FRET) measurements have been used to analyze fluorophore separations in a number of varying geometries, including small particles and extended surfaces. This study focuses on the geometry created by a donor extended above the surface of a small sphere (radius < R0), where the acceptors are integrated into the sphere surface. The model of this geometry was based on an amphipathic molecule with its lipophilic region integrated into a detergent micelle and its hydrophilic region extending outward from the micelle surface, where the donor fluorophore is attached to the hydrophilic region of the molecule. Based on random acceptor incorporation into the micelle, a Poisson distribution was used to calculate the distribution of acceptor probes across the micelle population. The model converges to RET on a flat surface when the radius of the micelle exceeds 0.8 R0. The model was also used to simulate FRET data showing that the positions of donors above the micelle surface could be uniquely resolved. Experimental verification of the model was achieved in a sulfobetaine palmitate micelle with fluorescein isothiocyanate donors attached to detergent-solubilized lipopolysaccharide (LPS) and lipophilic Fast-DiI acceptors. The use of steady-state analysis allowed resolution of cases in which donors were located at different distances from the surface. Combining steady-state with excited-state lifetime analysis allowed resolution of cases where there was a combination of distances. Given the large number of biomolecules that interact with lipids, this approach may prove generally useful for defining molecular conformation.

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