Thermomechanical transitions of egg-ceramide monolayers.

Ceramides have unique biophysical properties. Their high melting temperature and their ability to form lateral domains have converted ceramides into the paradigm of rigid lipids. Here, using shear surface rheology of egg-ceramide Langmuir monolayers, a solid to fluid transition was evidenced as a vanishing shear rigidity at lower temperatures than the lipid melting temperature. Such a mechanical transition, which depends on the lipid lateral pressure, was found in a broad range temperature (40-50 °C). The solid to fluid transition was correlated to a LC to LC+LE phase transition, as confirmed by BAM experiments. Interestingly, together with the softening transition, a supercooling process compatible with a glassy behavior was found upon freezing. A new phase scenario is then depicted that broadens the mechanical behavior of natural ceramides. The phase diversity of ceramides might have important implications in their physiological roles.

Fluorescent taxoids as probes of the microtubule cytoskeleton.

Microtubules are specifically and efficiently visualized with the new fluorescent taxoids 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol (FLUTAX) and 7-O-[N-(4'-tetramethylrhodaminecarbonyl)-L-alanyl]taxol (ROTAX). Similarly to taxol, FLUTAX and ROTAX are able to drive inactive GDP-liganded tubulin into microtubule assembly. One molecule of FLUTAX binds per alphabeta-tubulin dimer assembled, competing with taxol for the same microtubule binding site with an eightfold smaller relative affinity. FLUTAX-induced microtubule elongation is markedly Mg2+-dependent, encompassing the binding of one Mg2+ ion more per tubulin dimer polymerized than in the case of taxol. A small perturbation of the absorption spectrum of bound FLUTAX is consistent with a cationic microenvironment relative to the solution. The fluorescence anisotropy of FLUTAX increases by an order of magnitude upon binding to microtubules and time-resolved measurements indicate that the fluorescein moiety remains considerably mobile on a protein surface. The rate of labeling suggests that this is the outer microtubule wall. Alternatively, the microtubule lumen would be functional. FLUTAX- and ROTAX-induced microtubules, radial structures, and organized microtubule bundles are readily observed under the fluorescence microscope. Rapid and accurate visualization of native (or very mildly fixed) cytoplasmic and spindle microtubules of a variety of permeabilized cells is simply obtained with micromolar FLUTAX, with an advantage over immunofluorescence. In addition, FLUTAX labels the centrosomes of PtK2 cells more intensely than antibodies to alpha- or beta-tubulin, and co-localizing with antibodies to gamma-tubulin. Two brightly fluorescent spots, probably separating or duplicating centrioles, can be resolved in the centrosomes of interphase cells. This finding indicates that centrosomes may well be additional targets of action of taxoids. FLUTAX strongly labels microtubules near the spindle poles, as well as microtubules at the telophase spindle equator and the central part of the midbody in cytokinesis (instead of the dark zone frequently observed with immunofluorescence), suggesting a predominant interaction of FLUTAX with sites at which tubulin is newly polymerized. Nanomolar concentrations of FLUTAX also permit specific imaging of centrosomes, half-spindles and midbodies in growing U937 cells.

Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: a steady-state and time-resolved study of yeast phosphoglycerate kinase.

A multisite distance-based fluorescence resonance energy-transfer assay system was developed for the study of protein folding reactions. Single- and double-cysteine substitution mutagenesis was utilized to place sulfhydryl residues throughout the tertiary structure of the bidomain enzyme yeast phosphoglycerate kinase (PGK). These reactive cysteines were covalently modified with extrinsic donor [5-[[2-(2-iodoacetamido)ethyl]amino]-1-naphthalenesulfonic acid] and acceptor (5-iodoacetamidofluorescein) fluorescent labels. A detailed experimental strategy was followed, which revealed that, when these relatively large extrinsic fluorescent labels are covalently attached to properly selected solvent-exposed residues, they do not affect the intrinsic stability of the protein. The PGK crystal structure was combined with molecular dynamics simulations of the dyes built into the protein and time-resolved anisotropy experiments, in order to estimate a more realistic orientation factor, *, for each donor/acceptor pair. Time-resolved and steady-state fluorescence energy-transfer experiments revealed that this distance assay, spanning six different donor-acceptor distances, is linear and accurate (to within 10-20%) over the range of 30-70 A. This distance assay system for PGK allows for the measurement of long-range changes in intra- and interdomain spatial organization during protein folding reactions. The approach which we have developed can be applied to any protein system in which unique one- and two-site cysteine residues can be engineered into a protein. In the following paper [Lillo, M. P., et al. (1997) Biochemistry 36, 11273-11281], these multisite energy-transfer pairs are utilized for stopped-flow unfolding studies.

Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase.

Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics. If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the "second" genetic code problem would be solved. Regrettably, no such technique currently exists. As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Förster resonance energy transfer [Lillo, M. P., et al. (1997) Biochemistry 36, 11261-11272]. In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK. These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-[[[(2-iodoacetyl)amino]ethyl]amino]naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor). The unfolding of PGK is found to be a sequential multistep process (native --> I1 --> I2 --> unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded). Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1 and I2 states. With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A. As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study [Beechem, J. M., et al. (1995) Biochemistry 34, 13943-13948]. Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5-10 A.

Cholesterol effect on the physical state of lipid multibilayers from the platelet plasma membrane by time-resolved fluorescence.

There are indications that the plasma membrane lipid composition and, in particular, the cholesterol/phospholipid (C/PL) ratio, affects platelet function. As a first approximation to the molecular characterization of the effect of cholesterol on the order, fluidity and lateral heterogeneity of the platelet plasma membrane, the steady-state and time-resolved fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trans-parinaric acid (tPnA) has been studied in multibilayer vesicles of phospholipids extracted from human platelet plasma membrane with different cholesterol/phospholipid molar ratios modified in vitro from 0.07 to 0.9. The DPH studies show that the increased presence of cholesterol has a stronger effect on the order than on the fluidity of the bilayer, as has been previously observed in other lipid membranes. On the other hand, from the analysis of the fluorescence kinetics of tPnA we conclude that a higher cholesterol content gives rise to an increase of the heterogeneity of the bilayer, due to a larger fraction of solid-like lipid domains. These domains contain a cholesterol concentration much higher than the macroscopic average value.

Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments.

Multiphasic kinetics are often observed in stopped-flow investigations. To characterize further these kinetic phases, we have developed a methodology whereby fluorescence total intensity and anisotropy stopped-flow data can be combined in a single analysis. Fluorescence total intensity and anisotropy are highly interrelated and contain two very complementary forms of information. Total-intensity changes are useful in determining changes in populations with differing quantum yields, whereas anisotropy changes contain additional contributions caused by the rotational dynamics of the species. For cases in which the fluorescence quantum yield increases, the observed rate of anisotropy change will be more rapid than the total-intensity change, whereas in cases in which the total intensity decreases, the observed change in anisotropy will lag behind. In all cases, with quantum yield changes the stopped-flow anisotropy signals cannot be fit with models consisting of exponentials. Case studies examining these effects are described for the protein folding/refolding transitions of Staphylococcal nuclease and phosphoglycerate kinase. A multiphasic DNA exonuclease reaction using bacteriophage T4 DNA polymerase is also examined. In all of these cases, combined analysis of both data types revealed insights into reaction mechanism, which could not be obtained by either data type in isolation. Quantum yields and steady-state anisotropies associated with transiently populated intermediate species can be resolved. The data analysis methodologies described allow characterization of multiphasic reactions in terms of internally consistent kinetic rates, quantum yields, and steady-state anisotropies.

Conformation of human fibrinogen in solution from polarized triplet spectroscopy.

The rotational motions of human fibrinogen in solution at 20 degrees C have been examined, in the 0.2-12-microseconds time range, by measuring the laser-induced dichroism of the triplet state of an erythrosin probe covalently bonded to the protein. The decay of the anisotropy was multiexponential, and up to three correlation times (phi 1 = 380 +/- 50 ns, phi 2 = 1.1 +/- 0.1 microseconds, and phi 3 = 3.3 +/- 0.6 microseconds) were needed to obtain a satisfactory analysis. The experimental data are consistent with the brownian motions of an elongated, rigid particle. If the correlation times are combined with previous data on the intrinsic viscosity of fibrinogen, the rotational and translational diffusive properties of the protein can be reproduced with high accuracy by idealizing it as an elongated ellipsoid of revolution with dimensions (2a x 2b) of (54 +/- 6) x (7.2 +/- 0.5) nm, having rotational diffusion constants of D parallel = (6.2 +/- 0.7) x 10(5) s-1 and D perpendicular = (5 +/- 1) x 10(4) s-1. The possibility of Ca(2+)-dependent changes in the rigidity or conformation of fibrinogen was excluded by examining the submicrosecond time-resolved fluorescence depolarization of 1-methylpyrene conjugates of the protein in the presence of different calcium concentrations. Although there are inherent difficulties to extrapolate the data on isolated fibrinogen molecules to the polymerizing species, this relatively stiff conformation meets the requirements of the classical half-staggered double-stranded model of fibrin polymerization rather better than those of the recently proposed interlocked single-stranded mechanism.

Molecular order and fluidity of the plasma membrane of human platelets from time-resolved fluorescence depolarization.

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-[(4-trimethyl- amino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxy- ethyl)-1,6-diphenyl-1,3,5-hexatriene, 16(9-anthroyloxy)-palmitic acid, CIS-parinaric acid, trans-parinaric acid and perylene) to report changes induced by temperature and Ca2+ in the plasma membrane of human platelets has been examined. The steady-state fluorescence anisotropy of the probes was compared after being incorporated into whole resting platelets, fragments of platelet plasma membrane and multilayers of lipids extracted from these membranes. In addition, we have investigated the molecular order and dynamics of the three preparations by time-resolved fluorescence depolarization of DPH and CE-DPH as a function of temperature and Ca2+ concentration. The high values of the order parameters found in intact platelets (SDPH, 36 degrees C = 0.70) were almost identical to those in membrane fragments and lipid vesicles, suggesting that lipid-lipid interactions and, therefore, the lipid composition are the main factors influencing the probe order parameter. Other lipid interactions such as those with membrane proteins and intracellular components have little effect on the SDPH in platelets. These measurements also showed that the stationary fluorescence anisotropy of DPH and CE-DPH in platelets is largely determined (80%) by the structural order of the lipid bilayer. Therefore, the previous "microviscosity" values based on stationary anisotropy data reflect the alignment and packing rather than the mobility of the bilayer components. The dynamic component of the anisotropy decay of these probes was analyzed in terms of the wobbling-in-cone model, allowing an estimation of the apparent viscosity of platelet plasma membrane (eta DPH, 36 degrees C = 0.5 P) that is similar to that of the erythrocyte membrane. This value decreased substantially in multilayers of native lipids, indicating a large effect of the lipid-protein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25 degrees to 36 degrees C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36 degrees-40 degrees C interval. This may be related to the end-point of the lipid phase separation reported by Gordon et al. (1983). Finally, the rigidifying (lipid ordering) effect of Ca2+ on the platelet plasma membrane could also be observed by the fluorescence anisotropy measurements, in the form of an increase (approximately 2%) of the order parameter of CE-DPH for Ca2+ concentrations in the millimolar range.

Lateral heterogeneity in human platelet plasma membrane and lipids from the time-resolved fluorescence of trans-parinaric acid.

We have investigated the complex behaviour of the time resolved fluorescence intensity and anisotropy of trans-parinaric acid, incorporated into fragments of the plasma membrane of human platelets and in multibilayers of lipids extracted from that membrane. It is shown that the observation of anisotropies that increase at long times can be satisfactorily interpreted by assuming two populations of the fluorescence probe with distinct life-times, rotational relaxation times and order parameters. The heterogeneous probe distribution was correlated with a similar heterogeneity in the lipid composition of the bilayer, modulated by temperature. Below 35 degrees C an important fraction of the lipids of the plasma membrane are apparently in the form of solid-like domains (20% at 20 degrees C). However, in the physiological temperature range that solid/fluid heterogeneity is almost negligible. Since these effects were also observed in multibilayers of lipids from the platelet membrane, the formation of solid-like clusters appears to arise from lipid-lipid interactions only, and most probably involving cholesterol. These results support the previous finding of a lateral phase separation for temperatures less than 37 degrees C described by Gordon et al. (1983) in a spin-probe study of the platelet plasma membrane.

Protein structure probed by polarization spectroscopy. I. Evidence for fibrinogen rigidity from stationary fluorescence.

Two fluorescent derivatives of human fibrinogen have been synthesized, by the covalent bonding of 1-dimethylaminoaphthalene-5-sulphonyl and methylpyrene chromophores, to investigate the internal molecular dynamics of the protein in solution. The stationary fluorescence depolarization of these derivatives under isothermal conditions is described here while in an accompanying paper (part II) a time-resolved study is reported. From the static fluorescence data it is concluded that reorientational processes in the subnanosecond and microsecond time ranges account for all the observed depolarization. The faster motion was assigned to the restricted, localized oscillations of the label while the slow motion was ascribed to the overall rotation of the protein molecule. Consequently, the protein in solution appears considerably rigid in the 10-1000 ns range, in contrast with a previous conception of a flexible fibrinogen based on non-isothermal depolarization experiments. These previous experiments are, in fact, concordant with the rigid fibrinogen proposed here if they are reinterpreted using Weber's early ideas on thermally activated depolarization (G. Weber, J. Biochem. 51 (1952) 145).

Protein structure probed by polarization spectroscopy. II. A time-resolved fluorescence study of human fibrinogen.

Human fibrinogen in solution was studied by monitoring the time-resolved depolarization of the fluorescence emitted by two spectroscopic labels of which the fluorescence lifetimes differ by an order of magnitude. Contrary to a long-held view, no evidence of molecular flexibility was found in the 10-1000 ns range. In addition, from the rate of the overall rotation, it is proposed that a prolate and symmetric ellipsoid of 47 X 10.5 nm may represent the time-averaged hydrodynamic size and shape of the protein in solution. This rigid and highly hydrated structure (4 g water/g protein) accommodates the latest nodular models obtained from electron microscopy, explains the singular hydrodynamics of fibrinogen and, apparently, it would perform the two main functions of the protein in haemostasis, blood coagulation and platelet aggregation, more efficiently than the flexible molecule.