4-4-difluoro-4-bora-3a-4a-diaza-s-indacene and 1-palmitoyl-2-oleoylphosphatidylcholine

Phosphatidylinositol (4,5) bisphosphate (PIP2) is an important signaling molecule located on the inner leaflet of the cell membrane. In order to perform its various signaling functions, it is suggested that PIP2 must be able to form localized clusters. In this study, we have used LAURDAN generalized polarization function (GP) with unlabeled PIP2 and single point fluorescence correlation spectroscopy and brightness analysis of various BODIPY labeled PIP2 to determine the presence of clusters in the membrane of giant unilamellar vesicles (GUVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin and cholesterol. We determined the number of freely diffusing fluorescent BODIPY molecules in the membrane and found that in GUVs containing various amounts of labeled PIP2, this number was significantly lower than in GUVs made with the control BODIPY labeled hexadecyl phosphatidylcholine (BODIPY-HPC). Also, we noted an increase in brightness of the labeled PIP2 particles with increasing labeled PIP2 molar fraction. Together with the observed change in LAURDAN GP with increasing molar fraction of unlabeled PIP2, these results demonstrate the presence of PIP2 enriched clusters that are smaller than the resolution limit of the fluorescent microscope. In addition, we report the presence of a hypsochromic shift of the fluorescence for the BODIPY labeled lipids that we attributed to clustering. This clustering result in a change in the partitioning of the lipids with the BODIPY labeled PIP2 lipids able to move between the liquid ordered and liquid disordered phase.

Topics: Boron Compounds; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol 4,5-Diphosphate; Unilamellar Liposomes

In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tapping-mode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, which-depending on the concentration of the constituents-separates into liquid-disordered (l(d)), liquid-ordered (l(o)), and solid-ordered (s(o)) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is l(d) > l(o) >> s(o). Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the l(d)/l(o) phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.

Topics: Boron Compounds; Cholesterol; Fluorescent Dyes; Membrane Lipids; Membrane Microdomains; Microscopy, Atomic Force; Microscopy, Fluorescence; Models, Molecular; Phosphatidylcholines; ras Proteins; Sphingomyelins

Alpha-synuclein, a presynaptic protein associated with Parkinson's disease, is found as both soluble cytosolic and membrane-bound forms. Although the function of alpha-synuclein is unknown, several observations suggest that its association with membranes is important. In the present study we investigated the effect of alpha-synuclein on lipid oxidation in membranes containing phospholipids with unsaturated fatty acids. The kinetics of lipid oxidation were monitored by the change in fluorescence intensity of the dye C11-BODIPY. We find that monomeric alpha-synuclein efficiently prevented lipid oxidation, whereas fibrillar alpha-synuclein had no such effect. Our data suggest that the prevention of unsaturated lipid oxidation by alpha-synuclein requires that it bind to the lipid membrane. The antioxidant function of alpha-synuclein is attributed to its facile oxidation via the formation of methionine sulfoxide, as shown by mass spectrometry. These findings suggest that the inhibition of lipid oxidation by alpha-synuclein may be a physiological function of the protein.

Topics: alpha-Synuclein; Antioxidants; Boron Compounds; Circular Dichroism; Fluorescence; Kinetics; Liposomes; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylglycerols

The birefringence and linear dichroism of anisotropic thin films such as proteolipid membranes are related to molecular properties such as polarizability, shape, and orientation. Coupled plasmon-waveguide resonance (CPWR) spectroscopy is shown in the present work to provide a convenient means of evaluating these parameters in a single lipid bilayer. This is illustrated by using 1-10 mol % of an acyl chain chromophore-labeled phosphatidylcholine (PC) incorporated into a solid-supported PC bilayer deposited onto a hydrated silica surface. CPWR measurements were made of refractive index and extinction coefficient anisotropies with two exciting light wavelengths, one of which is absorbed by the chromophore and one of which is not. These results were used to calculate longitudinal and transverse molecular polarizabilities, the orientational order parameter and average angle between the longitudinal axis of the lipid molecule and the membrane normal, and the molecular shape factors of the lipid molecules. The values thereby obtained are in excellent agreement with parameters determined by other techniques, and provide a powerful tool for analyzing lipid-protein, protein-protein, and protein-ligand interactions in proteolipid films.

Topics: Anisotropy; Birefringence; Boron Compounds; Equipment Design; Fluorescent Dyes; Lipid Bilayers; Orientation; Phosphatidylcholines; Proteolipids; Surface Plasmon Resonance

Microsphere-based flow cytometric detection of cholera toxin (CT) through distance-dependent fluorescence resonant energy transfer (FRET) has been developed. Simultaneous double-fluorescence changes induced by multivalent interactions between CT and fluorophore (both fluorescence donor and acceptor)-labeled ganglioside GM1 on a biomimetic membrane surface (supported bilayers of phospholipids) can be measured by a commercial flow cytometer, providing a convenient and sensitive detection method for CT. The flow cytometry-based biosensor is capable of detecting less than 10 pM CT within 30 min. The signal generation strategy coupled with flow cytometry also provides a convenient method for kinetic studies of multivalent interactions. The surface density and the ratio of donor/acceptor-labeled GM1 on the surfaces of phospholipid bilayers are optimized to achieve high sensitivity.

Topics: Biosensing Techniques; Boron Compounds; Cholera Toxin; Flow Cytometry; Fluorescent Dyes; G(M1) Ganglioside; Humans; Kinetics; Lipid Bilayers; Phosphatidylcholines; Proteins; Sensitivity and Specificity; Serum Albumin; Time Factors

We determined the orientation of a biotinylated version of the pore-forming peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) at pH 5.0 in large unilamellar phosphatidylcholine vesicles, using the enhancement of BODIPY-avidin fluorescence subsequent to its irreversible binding to a biotin moiety. GALA and its variants were biotinylated at the N- or C-terminus. BODIPY-avidin was either added externally or was pre-encapsulated in vesicles to assess the fraction of liposome-bound biotinylated GALA that exposed its labeled terminus to the external or internal side of the bilayer, respectively. Under conditions where most of the membrane-bound peptides were involved in transmembrane aggregates and formed aqueous pores (at a lipid/bound peptide molar ratio of 2500/1), the head-to-tail (N- to C-terminus) orientation of the membrane-inserted peptides was such that 3/4 of the peptides exposed their N-terminus on the inside of the vesicle and their C-terminus on the outside. Under conditions resulting in reduced pore formation (at higher lipid/peptide molar ratios), we observed an increase in the fraction of GALA termini exposed to the outside of the vesicle. These results are consistent with a model (Parente et al., Biochemistry, 29:8720, 1990) that requires a critical number of peptides (M) in an aggregate to form a transbilayer structure. When the peptides form an aggregate of size i, with i < M = 4 to 6, the orientation of the peptides is mostly parallel to the membrane surface, such that both termini of the biotinylated peptide are exposed to external BODIPY-avidin. This BODIPY-avidin/biotin binding assay should be useful to determine the orientation of other membrane-interacting molecules.

Topics: Amino Acid Sequence; Avidin; Biophysical Phenomena; Biophysics; Biotin; Boron Compounds; Fluorescent Dyes; Hydrogen-Ion Concentration; Lipid Bilayers; Liposomes; Peptides; Phosphatidylcholines; Protein Binding

Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then incorporated into a biomimetic membrane surface. The presence of CT with five binding sites for GM1 causes dramatic change for the fluorescence of the labeled GM1. (1) In the scheme using fluorescence self-quenching as a signal-transduction mechanism, the fluorescence intensity drops significantly as a result of aggregation of the fluorophore-labeled GM1 on a biomimetic surface. (2) By labeling GM1 with a fluorescence energy transfer pair, aggregation of the labeled GM1 results in a decrease in donor fluorescence and an increase in acceptor fluorescence, providing a unique signature for selective protein-receptor binding. (3) In the third scheme, using the biomimetic surface as part of signal transduction and combining both fluorescence self-quenching and energy-transfer mechanisms to enhance the signal transduction, a signal amplification was achieved. The detection systems can reliably detect less than 0.05 nM CT with fast response (less than 5 min). This approach can easily be adapted to any biosensor scheme that relies on multiple receptors or co-receptors. The methods can also be applied to investigate the kinetics and thermodynamics of the multivalent interactions.

Topics: Albumins; Binding Sites; Boron Compounds; Cholera Toxin; Contrast Media; Energy Transfer; Fluorescein; Fluorescent Dyes; G(M1) Ganglioside; Lipid Bilayers; Membranes, Artificial; Optics and Photonics; Phosphatidylcholines; Proteins; Sensitivity and Specificity; Signal Transduction; Spectrometry, Fluorescence; Toxins, Biological