Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence

Parasassi, Tiziana; Stasió, Gelsomina De; Ravagnan, Giampietro; Rusch, Ruth M.; Gratton, Enrico

doi:10.1016/s0006-3495(91)82041-0

Cited by 810 publications

(991 citation statements)

References 26 publications

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“…Laurdan generalized polarization (GP) is a convenient probe for nanosecond relaxation changes correlated with the phase behavior of lipid bilayers [58][59][60] . From the results shown in Table 2 it can be seen that the parameters of the phase transition detected by this method are similar for Nanodiscs of different sizes.…”

Section: Discussionmentioning

confidence: 99%

Thermotropic Phase Transition in Soluble Nanoscale Lipid Bilayers

et al. 2005

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The role of lipid domain size and protein-lipid interfaces in the thermotropic phase transition of dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) bilayers in Nanodiscs was studied using small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and generalized polarization (GP) of the lipophilic probe Laurdan. Nanodiscs are watersoluble, monodisperse self-assembled lipid bilayers encompassed by a helical membrane scaffold protein (MSP). MSPs of different lengths were used to define the diameter of the Nanodisc lipid bilayer from 76 to 108 Å and the number of DPPC molecules from 164 to 335 per discoidal structure. In Nanodiscs of all sizes, the phase transitions were broader and shifted to higher temperatures relative to those observed in vesicle preparations. The size dependences of the transition enthalpies and structural parameters of Nanodiscs reveal the presence of a boundary lipid layer in contact with the scaffold protein encircling the perimeter of the disc. The thickness of this annular layer was estimated to be approximately 15 Å, or two lipid molecules. SAXS was used to measure the lateral thermal expansion of Nanodiscs and a steep decrease of bilayer thickness during the main lipid phase transition was observed. These results provide basis for the quantitative understanding of cooperative phase transitions in membrane bilayers in confined geometries at the nanoscale.

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Section: Discussionmentioning

confidence: 99%

Thermotropic Phase Transition in Soluble Nanoscale Lipid Bilayers

et al. 2005

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“…[1][2][3][4][5] It is critical to understand the photophysical properties of the fluorescent probe molecule, especially if it can emit from more than one state. 6-Propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) (Figure 1) is a highly fluorescent, hydrophobic molecule, first synthesized and characterized by Weber and Farris in 1979.…”

Section: Introductionmentioning

confidence: 99%

Solvation Dynamics of the Fluorescent Probe PRODAN in Heterogeneous Environments: Contributions from the Locally Excited and Charge-Transferred States

2009

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The coexistence of different excited states with different properties of the same chromophores could have significant consequences for the accurate characterization of solvation dynamics in a heterogeneous environment, such as a protein. The purpose of this work is to study the contributions of the locally excited (LE) and charge-transferred (CT) states of the fluorescent probe molecule 6-propionyl-2-(N,Ndimethylamino)naphthalene (PRODAN) to its solvation dynamics in the heterogeneous environment provided by reverse micelles formed by sodium 1,4-bis-(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane/ water. We have found that the LE and CT states of PRODAN solvate on different time scales in reverse micelles (2 and ∼0.4 ns, respectively), consistent with results suggested in the literature, and have concluded that PRODAN's use as a probe of heterogeneous environments must be used with caution and that, more importantly, the same caution must be exercised with any chromophore capable of emitting from different excited states. ReceiVed: June 1, 2009; ReVised Manuscript ReceiVed: July 20, 2009 The coexistence of different excited states with different properties of the same chromophores could have significant consequences for the accurate characterization of solvation dynamics in a heterogeneous environment, such as a protein. The purpose of this work is to study the contributions of the locally excited (LE) and charge-transferred (CT) states of the fluorescent probe molecule 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) to its solvation dynamics in the heterogeneous environment provided by reverse micelles formed by sodium 1,4-bis-(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane/water. We have found that the LE and CT states of PRODAN solvate on different time scales in reverse micelles (2 and ∼0.4 ns, respectively), consistent with results suggested in the literature, and have concluded that PRODAN's use as a probe of heterogeneous environments must be used with caution and that, more importantly, the same caution must be exercised with any chromophore capable of emitting from different excited states.

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“…Water penetration has been correlated with lipid packing and membrane fluidity. 18,19 A full discussion on the use and mathematical significance of GP can be found in the literature. 19,20 Other Techniques.…”

Section: Principlesmentioning

confidence: 99%

Lipid−Protein Interactions Revealed by Two-Photon Microscopy and Fluorescence Correlation Spectroscopy

Sánchez

Gratton

2005

Acc. Chem. Res.

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Cellular processes involve a multitude of chemical reactions that must be kept in delicate equilibrium to maintain cellular homeostasis. Powerful biophysical techniques are needed to measure the localization and concentration of target molecules as well as to quantify complex molecular processes in model and in vivo systems. Two-photon microscopy and fluorescence correlation spectroscopy (FCS) can measure association and dynamics of appropriate molecules under equilibrium conditions. FCS provides information on motility (diffusion coefficients), concentration (number of particles), association (molecular brightness), and localization (image) of the target molecules. All of this information, in conjunction with computational modeling techniques, can help us to better understand the network of complex molecular interactions, which are at the basis of cellular processes. Fluorescence imaging techniques add the beauty of visualization to the scientific information. Photons emitted by a fluorescent dye are digitized, and the associated spatial information and intensity can be translated into different colors and shades providing the researcher not only with quantitative intensity information but also with spatial resolution and visual comprehension of two-or threedimensional images. In this Account, we review the use of twophoton excitation microscopy and FCS in the study of lipidprotein interactions. We discuss these new methodologies and techniques, and we present examples of different complexity from qualitative to quantitative, from simple model systems to studies in living cells.

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Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence

Cited by 810 publications

References 26 publications

Thermotropic Phase Transition in Soluble Nanoscale Lipid Bilayers

Thermotropic Phase Transition in Soluble Nanoscale Lipid Bilayers

Solvation Dynamics of the Fluorescent Probe PRODAN in Heterogeneous Environments: Contributions from the Locally Excited and Charge-Transferred States

Lipid−Protein Interactions Revealed by Two-Photon Microscopy and Fluorescence Correlation Spectroscopy

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