Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Mutual effects of MinD-membrane interaction: I. Changes in the membrane properties induced by MinD binding

In Escherichia coli and other bacteria, MinD, along with MinE and MinC, rapidly oscillates from one pole of the cell to the other controlling the correct placement of the division septum. MinD binds to the membrane through its amphipathic C-terminal α-helix. This binding, promoted by ATP-induced dimerization, may be further enhanced by a consequent attraction of acidic phospholipids and formation of a stable proteolipid domain. In the context of this hypothesis we studied changes in dynamics of a model membrane caused by MinD binding using membrane-embedded fluorescent probes as reporters. A remarkable increase in membrane viscosity and order upon MinD binding to acidic phospholipids was evident from the pyrene and DPH fluorescence changes. This viscosity increase is cooperative with regards to the concentration of MinD-ATP, but not of the ADP form, indicative of dimerization. Moreover, similar changes in the membrane dynamics were demonstrated in the native inverted cytoplasmic membranes of E. coli, with a different depth effect. The mobility of pyrene-labeled phosphatidylglycerol indicated formation of acidic phospholipid-enriched domains in a mixed acidic-zwitterionic membrane at specific MinD/phospholipid ratios. A comparison between MinD from E. coli and Neisseria gonorrhea is also presented.     

Effect of structural modifications on the spectroscopic properties and dynamics of the excited states of peridinin

The spectroscopic properties and dynamics of the lowest excited singlet states of peridinin and two derivatives have been studied by steady-state absorption and fast-transient optical spectroscopic techniques. One derivative denoted PerOlEs, possesses a double bond and a methyl ester group instead of the r-ylidenebutenolide of peridinin. Another derivative denoted PerAcEs, is the biosynthetic precursor of peridinin and possesses a triple bond and a methyl ester group corresponding to the r-ylidenbutenolide function. Ultrafast time-resolved spectroscopic experiments in the visible and near-infrared regions were performed on the molecules and reveal the energies and regarding the structural features and interactions responsible for the unusual solvent-induced changes in the steady-state and transient absorption spectra and dynamics of dynamics of the excited electronic states. The data also provide information peridinin.     

Population shuffling between ground and high energy excited states

Stochastic processes powered by thermal energy lead to protein motions traversing time-scales from picoseconds to seconds. Fundamental to protein functionality is the utilization of these dynamics for tasks such as catalysis, folding, and allostery. A hierarchy of motion is hypothesized to connect and synergize fast and slow dynamics toward performing these essential activities. Population shuffling predicts a “top-down” temporal hierarchy, where slow time-scale conformational interconversion leads to a shuffling of the free energy landscape for fast time-scale events. Until now, population shuffling was only applied to interconverting ground states. Here, we extend the framework of population shuffling to be applicable for a system interconverting between low energy ground and high energy excited states, such as the SH3 domain mutants G48M and A39V/N53P/V55L from the Fyn tyrosine kinase, providing another tool for accessing the structural dynamics of high energy excited states. Our results indicate that the higher energy gauche− rotameric state for the leucine χ₂ dihedral angle contributes significantly to the distribution of rotameric states in both the major and minor forms of the SH3 domain. These findings are corroborated with unrestrained molecular dynamics (MD) simulations on both the major and minor states of the SH3 domain demonstrating high correlations between experimental and back-calculated leucine χ₂ rotameric populations. Taken together, we demonstrate how fast time-scale rotameric side-chain population distributions can be extracted from slow time-scale conformational exchange data further extending the scope and the applicability of the population shuffling model.     

Effects of fluorescent probe NBD-PC on the structure, dynamics and phase transition of DPPC. A molecular dynamics and differential scanning calorimetry study

We present a combined theoretical (molecular dynamics, MD) and experimental (differential scanning calorimetry, DSC) study of the effect of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl chain-labeled fluorescent phospholipid analogs (C6-NBD-PC and C12-NBD-PC) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. DSC measurements reveal that < 1 mol% of NBD-PC causes elimination of the pre-transition and a large loss of cooperativity of the main transition of DPPC. Labeling with C6-NBD-PC or C12-NBD-PC shifts the main transition temperature to lower or higher values, respectively. Following our recent report on the location and dynamics of these probes (BBA 1768 (2007) 467-478) in fluid phase DPPC, we present a detailed analysis of 100-ns MD simulations of systems containing either C6-NBD-PC or C12-NBD-PC, focused on their influence on several properties of the host bilayer. Whereas most monitored parameters are not severely affected for 1.6 mol% of probe, for the higher concentration studied (6.2 mol%) important differences are evident. In agreement with published reports, we observed that the average area per phospholipid molecule increases, whereas DPPC acyl chain order parameters decrease. Moreover, we predict that incorporation of NBD-PC should increase the electrostatic potential across the bilayer and, especially for C12-NBD-PC, slow lateral diffusion of DPPC molecules and rotational mobility of DPPC acyl chains.     

Interaction of liposomes with homologous series of fluorescent berberine derivatives: New cationic probes for measuring membrane potential

The interaction of liposomes with a series of fluorescent berberine derivatives having different alkyl chain lengths has been investigated. The hydrophobicity of the binding site on the phospholipid membrane increases and mobility decreases with the length of the alkyl chain. If lauryl sulphate micelles are used to bind berberines, the hydrophobicity of the binding site is the same for all derivatives. The dye series represents a model with constant charge and growing lipophilicity. Both electrostatic forces and lipophilicity play an important role in binding. By virtue of the excellent sensitivity of the dyes to medium polarity, berberines prove to be suitable probes for measuring membrane potential, but only in cases when a negative charge is generated in the liposomal interior. The fluorescence response is a linear function of the potential magnitude.     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

Setting up and running molecular dynamics simulations of membrane proteins

Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins.     

Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays

We describe a new procedure for the analysis of time-resolved decays of fluorescence intensity. This procedure was used to resolve the emission spectra of the initially excited and solvent relaxed states of a tryptophan derivative in viscous solution. Specifically, we examined N-acetyl-l-tryptophanamide (AcTrpNH₂) in viscous and nonviscous solutions of propylene glycol. Time-resolved decays of fluorescence intensity were collected at wavelengths across the emission spectra. Instead of the usual procedure of deconvolving these data with the time profile of the exciting pulse, we deconvolved these data using the response observed on the short-wavelength side of the emission. If one assumes that this emission results only from the initially excited state (F), then the nonzero decay time calculated using deconvolution is that of the solvent relaxed state (R). For our specific case of AcTrpNH₂ the emission spectra of the F and R states overlap at most wavelengths longer than the short-wavelength side of the emission (310 nm). As a result, differential-wavelength deconvolution yields two lifetimes and amplitudes, one pair representing the relaxed state and the other the initially excited state. The latter appears as a zero-decay-time component whose amplitude can be readily quantified. The wavelength-dependent amplitude of this zero-lifetime component can be used to calculate the emission spectrum of the F state and. by difference, the emission spectrum of the relaxed state. For AcTrpNH₂ in propylene glycol at ?20°C the emission maxima of the F and R states are near 320 and 350 nm, respectively, and the relative proportion of the emission from each state was near 50%. At lower temperatures the emission from the F state becomes dominant and at high temperatures the emission from the R state dominates. We note that this resolution of states is somewhat arbitrary because we assumed a two-state model and the absence of solvent relaxed emission at 310 nm. Nonetheless, differential-wavelength deconvolution simplifies and facilitates the analysis of time-resolved fluorescence data from samples which undergo excited state reactions. Moreover, this deconvolution procedure considerably simplifies the determination of the kinetic constants for reversible excited state reactions. The application of differential-wavelength deconvolution does not increase the time reqaired for data acquisition. This differential analysis procedure should enhance the usefulness and precision of pulse fluorometric methods in studies of nanosecond time scale processes in proteins and membranes.     

Solvent-assisted excited state proton transfer and photoacidity of 2-hydroxypyridine. A nonadiabatic dynamics study

Reactions involving proton transfer, especially those taking place in excited states (ESPT), received considerable attention. These reactions underlie many biological and biochemical processes that are vital to life. DNA mutations excited state funnelling and transport phenomena are just examples. Among many molecules undergoing this type of photoreactions, phenols show a unique property, the so-called photoacidity. In the present communication, solvent (methanol and ammonia) assisted excited state proton transfer and photo acidity of 2-hydroxypyridine (2HP) are investigated at the DFT M06-2X / def2pvv level of theory. Excited states of 2HP and its complexes with methanol and ammonia were examined at two levels. First, sampling a Wigner distribution of 300 points, the photoabsorption spectra were simulated within the nuclear ensemble approximation. Second, simulation of the dynamics of the excited states with the surface hopping method. Several separate spectral windows with three hundred trajectories each, were considered. Lifetime of excited states and photochemical channels observed were discussed.     

Photoacoustic studies of non-radiative relaxation of excited states in melanin

Photoacoustic measurements made at various chopping frequencies on dense acqueous melanin suspensions have revealed a significant dependence upon pH and redox state. It is shown that such behaviour, differing from the simple predictions of the Rosencwaig-Gersho theory, can be explained in terms of finite carrier diffusion and recombination times. The implications of these findings with respect to the amorphous semiconductor model proposed to describe the dynamic role of epidermal melanin are discussed. From the experimental data, values of physical parameters were calculated which allow a qualitative correlation between chemical states and electronic behaviour and, consequently, some aspects of the molecular biology of the melanosome, founded on a rigorous base.     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.     

爆炸波对生物膜微观创伤的分子动力学分析

爆炸冲击波作用到人体胸部时,肺部会出现肺出血及肺水肿等症状,这是人体爆炸创伤的主要原因,深入研究很有必要.为了更好地理解爆炸创伤的机理,应研究冲击波与微观组织作用的力学过程,但具有一定的难度.本文从基本的生物膜做起,运用分子动力学研究冲击波对DPPC膜造成的损伤,通过停止活塞来控制冲击波的冲量,观察冲击过程中膜的恢复情况.通过观察不同冲量下冲击波经过膜后磷脂分子及其周围水分子分布,发现随着冲量增大,膜越来越无序混乱,褶皱更严重,疏水区水分子越来越多.将膜冲击过程划为3个阶段,分别为冲击阶段、恢复阶段和后效阶段.发现当冲量大于153 m Pa·s时,在冲击过程中没有观察到膜的损伤恢复.     

Effects of deformability and thermal motion of lipid membrane on electroporation: by molecular dynamics simulations

Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0 kcal/(mol Ų) in the external electric field of 1.4 kcal/(mol Å e), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2 kcal/(mol Ų) in the position constraints on lipid tails in the external electric field of 2.0 kcal/(mol Å e), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease.     

Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping,deplete cellular ATP and inhibit respiration of isolated mitochondria

3,3′-Dipropylthiodicarbocyanine iodide, a widely used fluorescent probe of membrane potential, was found to inhibit anti-Ig antibody, induced capping of mouse lymphocytes. The dye also lowered the cell ATP content. Experiments with isolated mitochondria revealed that the probe had a potent inhibitory action at site I of the respiratory chain. This mitochondrial blockade helps to explain the ATP depletion and blockade of capping, and gives cause for caution in the use of this dye as a probe of cell membrane potential.Three related dicarbocyanine dyes had similar toxic effects, but two cyanine dyes with much longer alkyl side chains, which have been used as probes of membrane fluidity, did not.     

Markov state modeling of membrane transport proteins

The flux of ions and molecules in and out of the cell is vital for maintaining the basis of various biological processes. The permeation of substrates across the cellular membrane is mediated through the function of specialized integral membrane proteins commonly known as membrane transporters. These proteins undergo a series of structural rearrangements that allow a primary substrate binding site to be accessed from either side of the membrane at a given time. Structural insights provided by experimentally resolved structures of membrane transporters have aided in the biophysical characterization of these important molecular drug targets. However, characterizing the transitions between conformational states remains challenging to achieve both experimentally and computationally. Though molecular dynamics simulations are a powerful approach to provide atomistic resolution of protein dynamics, a recurring challenge is its ability to efficiently obtain relevant timescales of large conformational transitions as exhibited in transporters. One approach to overcome this difficulty is to adaptively guide the simulation to favor exploration of the conformational landscape, otherwise known as adaptive sampling. Furthermore, such sampling is greatly benefited by the statistical analysis of Markov state models. Historically, the use of Markov state models has been effective in quantifying slow dynamics or long timescale behaviors such as protein folding. Here, we review recent implementations of adaptive sampling and Markov state models to not only address current limitations of molecular dynamics simulations, but to also highlight how Markov state modeling can be applied to investigate the structure–function mechanisms of large, complex membrane transporters.     

Higher-order polarizabilities of polymethine systems in ground and excited electronic states

The electronic higher-order polarizabilities of linear and cyclic polymethine systems withelectron donor and acceptor groups included in the conjugation systems, in the ground and first excited singlet and triplet states, are studied using semiempirical quantum-chemical calculations (MNDO and PPP-DCI). It is shown that these polarizabilities are determined by two main factors: the bond order alternation in the conjugated system and the magnitude of the electron transfer within the molecule. The effect oftrans-cis isomerisation of the linear polymethines is also studied.     

Kinetics of the rearrangement of the solvation shell of an excited fluorescent probe 4″-dimethylaminochalcone

A study was made of the processes associated with the quenching of 4″-dimethylaminochalcone (DMAC) fluorescence by proton-donor solvent (1-butanol). The kinetics of deactivation of the DMAC excited state was assessed by transient absorption spectra with a time resolution about 50 fs and by fluorescence decay with ～30-ps resolution. The following sequence of events could thus be envisaged: (i) the DMAC molecule in the ground state (prior to excitation) makes a hydrogen bond with an alcohol molecule; (ii) absorption of a light quantum causes a corresponding increase of the DMAC dipole moment; the H-bond is retained; (iii) the solvation shell formed by alcohol dipoles is reorganized in response to the raise of the DMAC dipole moment, with an energy expenditure about 24 kJ/mol and a time constant about 40 ps; the initial H-bond is still retained; (iv) processes leading to fluorescence quenching occur with an effective time constant of nearly 200 ps. Since quenching is far slower than solvate rearrangement, one can suppose that it is not a direct consequence of shell relaxation or prior H-bonding. Thus, DMAC fluorescence quenching may involve different processes observed with other aromatic molecules: H-bond rearrangement from a nonquenching to a more ‘efficient’ conformation, charge transfer between the excited molecule and alcohol, or solvent-induced out-of-plane twist of the DMAC amino group.