Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes

p13suc1 acts in the fission yeast cell division cycle as a component of p34 cdc2 . In the present work, structural information contained in the intrinsic fluorescence of p13 suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13 suc1 is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained with p13 suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13 suc1 is best described by three discrete lifetimes of 0.6 ns ( 1 ), 2.9 ns ( 2 ), and 6.1 ns (

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“…According to this method, the fitting functions to obtain r(t) are the following (Ameloot et al, 1984;Cross and Fleming, 1984),…”

Section: Methodsmentioning

confidence: 99%

Intrinsic Fluorescence Properties and Structural Analysis of p13 from Schizosaccharomyces pombe

Neyroz

Menna

Polverini

et al. 1996

Journal of Biological Chemistry

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“…In view of the approximate cylindrical shape of the DPH-propionic acid probe, its rotational diffusion in the membrane is assumed to be axially symmetric with a parallel (D II) and perpendicular (D±) diffusion coefficient. Because D± is much smaller than D II, for the analysis of the fluorescence experiments only D 1 needs to be taken into account (Ameloot et al 1984). The initial anisotropy, r (0), depends on the angle between the absorption and emission moments.…”

Section: Methodsmentioning

confidence: 99%

“…The initial anisotropy, r (0), depends on the angle between the absorption and emission moments. This parameter was fixed to a value of 0.39 as has been obtained by other authors (Ameloot et al 1984;Pottel et al 1986). …”

Section: Methodsmentioning

confidence: 99%

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

et al. 1992

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Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the alpha-helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to beta-polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D parallel) and perpendicular (D perpendicular) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P infinity to L/P 15 results in a decrease of the rotational diffusion coefficient D perpendicular from 3.4 x 10(7) to 1.9 x 10(7) s-1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters and , and the rotational diffusion coefficient D perpendicular of the fluorescent probe. The order parameters and increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…From the experimental anisotropy decay function, and applying the rotational diffusion model to rotation around an axis perpendicular to the symmetry axis of rod-like probes such as DPH or TMA-DPH [11,83], the rotational diffusion coefficient D ⊥ may be calculated as:…”

Section: Probe Rotational and Lateral Diffusionmentioning

confidence: 99%

“…Engel and Prendergast [10] observed a significantly higher value of long-time residual anisotropy for TMA-DPH compared to DPH in fluid bilayers, denoting a much more restricted degree of rotational motion for the former, consistent with its location in a more ordered environment. Bimodal angular distributions of the long molecular axis, with a maximum parallel and the other perpendicular (in the bilayer midplane, between the two monolayer leaflets) to the bilayer normal, have been proposed for DPH, but not for TMA-DPH, whose depolarization could be explained without invoking a population of probe with orientation along the bilayer plane [11][12][13]. On the other hand, quenching of DPH and TMA-DPH fluorescence by a series of nitroxidelabeled lipids was used to estimate the depth of fluorophore location in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) fluid vesicles, in absence and presence of cholesterol, using the parallax method [14].…”

Section: Introductionmentioning

confidence: 99%

Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

Canto

Robalo

Santos

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

102

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Fluorescence spectroscopy and microscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in these membranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only~3-4 Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using them as membrane reporters.

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Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes

Cited by 134 publications

References 32 publications

Intrinsic Fluorescence Properties and Structural Analysis of p13 from Schizosaccharomyces pombe

Intrinsic Fluorescence Properties and Structural Analysis of p13 from Schizosaccharomyces pombe

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

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