Solution and Interface Aggregation States of Crotalus atrox Venom Phospholipase A₂ by Two-Photon Excitation Fluorescence Correlation Spectroscopy

Abstract

The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.

Interaction of high density lipoprotein particles with membranes containing cholesterol

“…Experimental autocorrelation functions were fit using a Gaussian-Lorentzian intensity profile model, as described previously (24,37), which contains the formulas for the point-spread function and the definition of the beam waist used. The beam waist of the excitation profile function depends on the instrument setup and must be calibrated each time the system is aligned; for this purpose, fluorescein was used.…”

“…GUVs constitute an ideal model for microscopic studies because of their size (20-50 mm); this fact permits the direct observation of micrometer-scale domains that can be observed directly by fluorescence microscopy and the study of liquid phases from a more controlled physical perspective than is possible in cells. The GUVs have been used extensively in different studies (18)(19)(20)(21)(22)(23), and we have used them to study the interaction of phospholipase A 2 (24), apoA-I, and rHDL with lipid bilayers (17,25,26). We used LAURDAN generalized polarization (GP) imaging to characterize phase organization in GUVs made of different lipid mixtures containing cholesterol, and we show the usefulness of this technique to quantify the kinetics of cholesterol removal from different membrane pools.…”

Direct Evidence That Neural Cell Adhesion Molecule (NCAM) Polysialylation Increases Intermembrane Repulsion and Abrogates Adhesion

“…The fluorescence intensity was determined by averaging the intensities from a minimum of 10 images taken from different regions of the supported bilayer. where G() is the autocorrelation amplitude and ␦F(t) is the fluctuation in the number of molecules in the excitation volume at time t (25,26). If applied to the translational diffusion of point-like particles, Equation 2 can be expressed as the following.…”

“…The protein was separated from excess dye by NTA affinity chromatography as described above, and was stored at 4°C until use. The instrumentation for the measurement and the software for the analysis of FCS data are at the Laboratory for Fluorescence Dynamics at the University of Illinois at Urbana-Champaign (24,26). To excite the sample, a mode-locked Ti:sapphire laser (Mira 900, Coherent, Palo Alto, CA) pumped by an intracavity doubled Nd:YVO4 vanadate laser (Verdi, Coherent Inc., Santa Clara, CA) was used as a photon source.…”

“…A Zeiss Axiovert 135 TV microscope (Thornwood, NY) was used with an oil immersion objective (NA ϭ 1.4). The PSA-NCAM and NCAM concentrations used in the FCS experiments were ϳ10 Ϫ8 M. Data were analyzed to determine the diffusion coefficient by fitting the experimental results to an autocorrelation function that assumes a Gaussian-Lorentzian intensity profile (25,26). The Gaussian-Lorentzian function depends on the experimental setup and therefore must be calibrated.…”