Quantitative characterization of membrane-protein reversible association using FCS.

Functionally meaningful reversible protein-membrane interactions mediate many biological events. Fluorescence correlation spectroscopy (FCS) is increasingly used to quantitatively study the non-reversible binding of proteins to membranes using lipid vesicles in solution. However, the lack of a complete description of the phase and statistical equilibria in the case of reversible protein-membrane partitioning has hampered the application of FCS to quantify the partition coefficient (Kx). In this work, we further extend the theory that describes membrane-protein partitioning to account for spontaneous protein-membrane dissociation and reassociation to the same or a different lipid vesicle. We derive the probability distribution of proteins on lipid vesicles for reversible binding and demonstrate that FCS is a suitable technique for accurate Kx quantification of membrane-protein reversible association. We also establish the limits to Kx determination by FCS studying the Cramer-Rao bound on the variance of the retrieved parameters. We validate the mathematical formulation against reaction-diffusion simulations to study phase and statistical equilibria and compare the Kx obtained from a computational FCS titration experiment with the experimental ground truth. Finally, we demonstrate the application of our methodology studying the association of anti-HIV broadly neutralizing antibody (10E8-3R) to the membrane.

Fast Hydrolysis and Strongly Basic Water Adducts Lead to Potent Os(II) Half-Sandwich Anticancer Complexes.

Complexes of the formula [Os(η6-arene)(C,N-phenylpyridine)Z] (where Z is chlorido or a tethered oxygen) undergo very fast Os-Z hydrolysis (<5 min), and the high basicity of the coordinated water molecule of the aqua adducts (Os-OH2; pKa > 8) very much contrasts with previously reported Os-aqua adducts bearing NN- and NO-chelating ligands (pKa < 6). The Os-Cl bond is unreactive in pure DMSO, yet the complexes readily form DMSO adducts upon aquation when dimethyl sulfoxide is present. Such a peculiar aqueous behavior is directly related to the negatively charged CN ligand. Potent Os-CN compounds (but not their Os-NN analogues) are particularly reactive; they bind to cysteine in vitro and decrease the activity of thioredoxin reductase (TrxR) in living cancer cells. By revealing some interesting structure-activity relationship on Os-CN vs Os-NN complexes, we start uncovering the molecular rationale for the successful biological applications of osmium(II) half-sandwich compounds.

A new interpretation of the absorption and the dual fluorescence of Prodan in solution.

Remarkable interest is associated with the interpretation of the Prodan fluorescent spectrum. A sequential hybrid Quantum Mechanics/Molecular Mechanics method was used to establish that the fluorescent emission occurs from two different excited states, resulting in a broad asymmetric emission spectrum. The absorption spectra in several solvents were measured and calculated using different theoretical models presenting excellent agreement. All theoretical models [semiempirical, time dependent density functional theory and and second-order multiconfigurational perturbation theory] agree that the first observed band at the absorption spectrum in solution is composed of three electronic excitations very close in energy. Then, the electronic excitation around 340 nm-360 nm may populate the first three excited states (π-π*Lb, n-π*, and π-π*La). The ground state S0 and the first three excited states were analyzed using multi-configurational calculations. The corresponding equilibrium geometries are all planar in vacuum. Considering the solvent effects in the electronic structure of the solute and in the solvent relaxation around the solute, it was identified that these three excited states can change the relative order depending on the solvent polarity, and following the minimum path energy, internal conversions may occur. A consistent explanation of the experimental data is obtained with the conclusive interpretation that the two bands observed in the fluorescent spectrum of Prodan, in several solvents, are due to the emission from two independent states. Our results indicate that these are the n-π* S2 state with a small dipole moment at a lower emission energy and the π-π*Lb S1 state with large dipole moment at a higher emission energy.

New insights on the fluorescent emission spectra of Prodan and Laurdan.

Prodan and Laurdan are fluorescent probes largely used in biological systems. They were synthetized to be sensitive to the environment polarity, and their fluorescent emission spectrum shifts around 120 nm, from cyclohexane to water. Although accepted that their emission spectrum is composed by two emission bands, the origin of these two bands is still a matter of discussion. Here we analyze the fluorescent spectra of Prodan and Laurdan in solvents of different polarities, both by decomposing the spectrum into two Gaussian bands and by computing the Decay Associated Spectra (DAS), the latter with time resolved fluorescence. Our data show that the intensity of the lower energy emission band of Prodan and Laurdan (attributed, in the literature, to the decay of a solvent relaxed state) is higher in cyclohexane than in water, showing a decrease as the polarity of the medium increases. Moreover, in all solvents studied here, the balance between the two emission bands is not dependent on the temperature, strongly suggesting two independent excited states. Both bands were found to display a red shift as the medium polarity increases. We propose here a new interpretation for the two emission bands of Prodan and Laurdan in homogeneous solvents: they would be related to the emission of two independent states, and not to a pair of non-relaxed and solvent relaxed states.

Electric dipole moments of the fluorescent probes Prodan and Laurdan: experimental and theoretical evaluations.

Several experimental and theoretical approaches can be used for a comprehensive understanding of solvent effects on the electronic structure of solutes. In this review, we revisit the influence of solvents on the electronic structure of the fluorescent probes Prodan and Laurdan, focusing on their electric dipole moments. These biologically used probes were synthesized to be sensitive to the environment polarity. However, their solvent-dependent electronic structures are still a matter of discussion in the literature. The absorption and emission spectra of Prodan and Laurdan in different solvents indicate that the two probes have very similar electronic structures in both the ground and excited states. Theoretical calculations confirm that their electronic ground states are very much alike. In this review, we discuss the electric dipole moments of the ground and excited states calculated using the widely applied Lippert-Mataga equation, using both spherical and spheroid prolate cavities for the solute. The dimensions of the cavity were found to be crucial for the calculated dipole moments. These values are compared to those obtained by quantum mechanics calculations, considering Prodan in vacuum, in a polarizable continuum solvent, and using a hybrid quantum mechanics-molecular mechanics methodology. Based on the theoretical approaches it is evident that the Prodan dipole moment can change even in the absence of solute-solvent-specific interactions, which is not taken into consideration with the experimental Lippert-Mataga method. Moreover, in water, for electric dipole moment calculations, it is fundamental to consider hydrogen-bonded molecules.

Optical characterization of Prodan aggregates in water medium.

The fluorescent probe Prodan (2-dimethylamino-6-propionylnaphthalene) has been widely used in biological systems, mainly due to the high sensitivity of its emission spectrum to the medium polarity. Though mostly used as a membrane probe, in lipid dispersions Prodan partitions in water, mainly in the presence of gel-phase bilayers. Here, optical properties of Prodan in aqueous medium are experimentally studied using absorption and emission spectroscopies, and compared with those of the probe in cyclohexane, where it is supposed to be very soluble. In parallel, theoretical calculations of the absorption spectrum of a monomer and aggregated Prodan in water were performed. Moreover, to understand Prodan-water and Prodan-Prodan interactions, solvation free energies of Prodan in water and in liquid Prodan were calculated. A light scattering profile underneath the optical absorption spectrum of Prodan in water clearly indicates the presence of aggregates at very low Prodan concentrations (0.9 µM). Experimental evidence of Prodan aggregation is theoretically supported by solvation free energy calculations, which demonstrate that Prodan molecules interact preferentially with other Prodan molecules than with water molecules. Theoretical calculations for electronic transition energies of monomers and aggregated Prodan in water show that a Prodan optical absorption band at 358 nm is related to the monomeric form of Prodan. This band saturates as Prodan concentration increases, indicating that aggregated Prodan prevails at higher concentrations. The relative increase in Prodan aggregated population is monitored by the increase in an absorption band at higher energies, at around 250 nm, and by the disappearance of a band at around 280 nm. Surprisingly, it was observed that the fluorescent emission spectrum of Prodan is not sensitive to probe aggregation up to around 15 µM. Hence, Prodan aggregation in water medium, even at very low concentrations, must be considered when using this fluorescent probe in biological systems, having in mind that its fluorescence spectrum is rather insensitive to aggregation.

The new fluorescent membrane probe Ahba: a comparative study with the largely used Laurdan.

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.

Molecular dynamics investigations of PRODAN in a DLPC bilayer.

Molecular dynamics computer simulations have been performed to identify preferred positions of the fluorescent probe PRODAN in a fully hydrated DLPC bilayer in the fluid phase. In addition to the intramolecular charge-transfer first vertical excited state, we considered different charge distributions for the electronic ground state of the PRODAN molecule by distinct atomic charge models corresponding to the probe molecule in vacuum as well as polarized in a weak and a strong dielectric solvent (cyclohexane and water). Independent on the charge distribution model of PRODAN, we observed a preferential orientation of this molecule in the bilayer with the dimethylamino group pointing toward the membrane's center and the carbonyl oxygen toward the membrane's interface. However, changing the charge distribution model of PRODAN, independent of its initial position in the equilibrated DLPC membrane, we observed different preferential positions. For the ground state representation without polarization and the in-cyclohexane polarization, the probe maintains its position close to the membrane's center. Considering the in-water polarization model, the probe approaches more of the polar headgroup region of the bilayer, with a strong structural correlation with the choline group, exposing its oxygen atom to water molecules. PRODAN's representation of the first vertical excited state with the in-water polarization also approaches the polar region of the membrane with the oxygen atom exposed to the bilayer's hydration shell. However, this model presents a stronger structural correlation with the phosphate groups than the ground state. Therefore, we conclude that the orientation of the PRODAN molecule inside the DLPC membrane is well-defined, but its position is very sensitive to the effect of the medium polarization included here by different models for the atomic charge distribution of the probe.

Study of blood porphyrin spectral profile for diagnosis of chronic renal failure.

The progression to end-stage renal failure is independent of the initial pathogenic mechanism. Metabolic acidosis is a common consequence of chronic renal failure that results from inadequate ammonium excretion and decreased tubular bicarbonate reabsorption. Protoporphyrin IX (PpIX) is the immediate metabolic precursor of the heme molecule. The purpose of this study was to evaluate the levels of erythrocytes protoporphyrin IX at an animal model during progressive renal disease. A total of 36 eight-week-old male Wistar rats were divided into six groups: Normal, 4 and 8 weeks after 5/6 nephrectomy (NX). Renal function was evaluated by creatinine clearance and plasma creatinine levels. The autofluorescence of erythrocytes porphyrin of healthy and NX rats was analyzed using fluorescence spectroscopy. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and NX rats autofluorescence shape occurred in the 600-700 nm spectral region. A correlation was observed between emission band intensity at 635 nm and progression of renal disease.

Vesicles with charged domains.

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.

Laurdan spectrum decomposition as a tool for the analysis of surface bilayer structure and polarity: a study with DMPG, peptides and cholesterol.

The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone alpha-MSH (Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2)) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly decreases the bilayer surface hydration.

Lipid bilayer pre-transition as the beginning of the melting process.

We investigate the bilayer pre-transition exhibited by some lipids at temperatures below their main phase transition, and which is generally associated to the formation of periodic ripples in the membrane. Experimentally we focus on the anionic lipid dipalmytoylphosphatidylglycerol (DPPG) at different ionic strengths, and on the neutral lipid dipalmytoylphosphatidylcholine (DPPC). From the analysis of differential scanning calorimetry traces of the two lipids we find that both pre- and main transitions are part of the same melting process. Electron spin resonance of spin labels and excitation generalized polarization of Laurdan reveal the coexistence of gel and fluid domains at temperatures between the pre- and main transitions of both lipids, reinforcing the first finding. Also, the melting process of DPPG at low ionic strength is found to be less cooperative than that of DPPC. From the theoretical side, we introduce a statistical model in which a next-nearest-neighbor competing interaction is added to the usual two-state model. For the first time, modulated phases (ordered and disordered lipids periodically aligned) emerge between the gel and fluid phases as a natural consequence of the competition between lipid-lipid interactions.

Laurdan in fluid bilayers: position and structural sensitivity.

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41 degrees C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel-fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.