Jean-Claude Brochon

Lipid clustering in bilayers detected by the fluorescence kinetics and anisotropy of trans-parinaric acid.

Fluid heterogeneity in lipid bilayers and shows a simple and useful method to quantify this heterogeneity. Taking advantage of the maximum entropy method, we have resolved the probe fluorescence lifetime distributions in homogeneous solutions and in single and two-component lipid bilayers at different temperatures. A precise description of the emission kinetics was obtained as a function of viscosity in the homogeneous solution and as a function of the phase composition (gel/fluid) in the lipid bilayers. These data show, unambiguously, that the same distribution pattern, with two well resolved lifetime classes, is observed both in pure solvents and in fluid bilayers. This distribution is modified during the thermotropic phase transition, with the appearance of a long lifetime component. The anisotropy experiments confirm that the amplitude of this component is proportional to the fraction of probe located in the gel phase. From this fraction we have quantified the amount of gel phase in the binary bilayer system dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine and determined the thermotropic phase diagram of the mixture. This phase diagram agrees well with that calculated assuming ideal mixing of the lipids (Marbrey, S., and J.M. Sturtevant. 1976. Proc. Natl. Acad. Sci. USA. 73:862-3866).

Data analysis in frequency-domain fluorometry by the maximum entropy method — recovery of fluorescence lifetime distributions

Abstract The maximum entropy method has been applied to the analysis of data obtained from multifrequency phase-modulation fluorometry. Analysis of real data (mixtures of dyes, dye embedded in a rigid matrix) and simulated data (broad lifetime distribution) are presented for illustrating the efficiency of the method.

HIV-1 Integrase Catalytic Core: Molecular Dynamics and Simulated Fluorescence Decays

Two molecular dynamics simulations have been carried out on the HIV-1 integrase catalytic core starting from fully determined crystal structures. During the first one, performed in the absence of divalent cation (6-ns long), the catalytic core took on two main conformations. The conformational transition occurs at approximately 3.4 ns. In contrast, during the second one, in the presence of Mg(2+) (4-ns long), there were no such changes. The molecular dynamics simulations were used to compute the fluorescence intensity decays emitted by the four tryptophan residues considered as the only chromophores. The decay was computed by following, frame by frame, the amount of chromophores that remained excited at a certain time after light absorption. The simulation took into account the quenching through electron transfer to the peptide bond and the fluorescence resonance energy transfer between the chromophores. The fit to the experimental intensity decays obtained at 5 degrees C and at 30 degrees C is very good. The fluorescence anisotropy decays were also simulated. Interestingly, the fit to the experimental anisotropy decay was excellent at 5 degrees C and rather poor at 30 degrees C. Various hypotheses such as dimerization and abnormal increase of uncorrelated internal motions are discussed.

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.

Maximum entropy analysis of dynamic parameters via the Laplace transform

Research in the field of Brownian motion often raises the problem of inverting the Laplace transform. Thus, the measured multi-exponential relaxations are the Laplace transform of the spectrum of decay times. However, the inversion of the Laplace transform is very ill-conditioned. The maximum entropy (maxent) method is shown to be a very powerful technique for such data analysis, capable of handling single peaks, multiple peaks and broad distributions in the spectra.After a theoretical demonstration of why entropy chooses the optimal reconstruction, the power of the technique is illustrated with two types of experimental examples related to Brownian dynamics. In the first example, photon correlation spectroscopy (PCS) is applied to concentrated colloidal dispersions. The results, analysed by maxent, present a detailed and coherent picture of the dynamics of a concentrated colloidal solution, leading to a test of the many-body hydrodynamic theories. In the second example, the maxent analysis of the time-resolved fluorescent decay of L-tryptophan is presented. Maxent provides some evidence that one of the features is broadened, suggestive of translative motion in the structure and/or heterogeneity of the fluorophores environment.

Time-correlated models applicable to reactions in restricted geometries: phospholipid vesicles in their gel and liquid crystalline phases

Abstract The flourescence decay profiles recorded with pyrene and with the florescence quenching process between diphenylhexatriene and 12-doxylstearic acid methyl ester in small unilamellar vesicles of dipalmitoylphos-phatidylglycerol and l -α-phosphatidyglycerol have been analyzed with a model appropriate for reactions in restricted geometries. Measurements were made both in the gel and in the liquid crystalline phase, either by changing temperature or by changing the composition of a phospholipid mixture. We have found that in the gel phase, the reactions are more restricted, that is, they proceed more locally than in the liquid crystalline phase. The number of solubilization sites available to the reactants is much larger in the liquid crystalline phase than in the gel phase for pyrene, and it is much larger, tending to infinity, for the diphenylhexatriene quencher couple. The probability of an immediate encounter between two reactants decreases from the gel to the liquid crystalline phase and as the temperature increases. In a short time scale of observation, the rate of the diffusion-controlled reaction increases with temperatur phase. However, when the time scale is large, long-distance diffusion, even in the liquid crystal-line phase and at high temperature, is impossible.