Session W47: Membranes: Biological and Synthetic

Permeation of anions through the central pore of human aquaporin 5

Aquaporin 5 (AQP5) plays an essential role in the physiology of saliva, tears and pulmonary secretion. We performed in silico experiments of the L51R mutant of the human Aquaporin 5 (PDB code: 3D9S) for which in vitro experimental data became available recently. Molecular Dynamic Simulations performed on the AQP5 tetramer embedded in a lipid bilayer reveal that the central pore of the AQP5 mutant lost the hydrophobicity of the wild type protein and becomes permeable to anions, but not to cations. This conclusion is in agreement with the in vitro experiments of Qin and Baron, 2013. Quantitatively, we compute the potential of mean force (PMF) of chloride and iodine anions along the permeation path through the central pore. We correlate the PMFs with the experimentally measured conductance of various anions, elucidating the atomistic details of ion conduction through AQP5 mutant.   

Discovery of a fundamental force related to a membrane's order-disorder transition that can govern protein self-assembly in membranes

The clustering of proteins in cell membranes is a controlling factor in biological processes such as cell signaling and membrane fusion. Using large-scale molecular simulations and a theoretical framework inspired by modern theories of the hydrophobic effect, we have uncovered a fundamental physical force for assembly of trans-membrane proteins in lipid bilayers. This force is a mesoscopic manifestation of the transition between ordered (i.e., gel) and disordered (i.e., fluid) phases of lipid bilayers. It is a pre-transition effect, occurring below the order-disorder transition temperature, nucleated by the protein's disturbance of the ordered phase. This powerful force acts over several nanometers in range. Conditions at which this force occurs and can lead to clustering of proteins in cell membranes will be discussed.   

Phase Behavior in Free and Supported Lipid Bilayers

Lipid bilayer structures supported on solid substrates are highly important experimental analogs for understanding cell membranes. In this study we use MARTINI coarse-grained force field to systematically investigate differences in phase behavior on supported and free membranes expanding our preliminary work [1]. Our results show that the same phases are found in both cases but that the phase boundaries are different both in temperature and concentration. We also find differences in the structure of the membranes. We particularly study the effect of cholesterol in supported bilayers for the first time. \\[4pt] [1] C. Xing, R. Faller J Phys Chem B 2008.   

Theory for registered and antiregistered phase separation of amphiphilic bilayers

Phase separation in bilayers can be exploited by nature, and engineers, to design-in function via membrane domains. The presence of two separate, yet coupled, leaflets forces one to ask whether and how such domains are aligned (registered) across the bilayer. Experiment and simulation yield intriguingly disparate observations. We introduce a theory for phase separation in coupled leaflets, by explicitly coarse-graining a lattice model that includes molecule-level structure and interactions. We show that hydrophobic mismatch leads to a complex competition of inter-leaflet couplings. The theory helps unify prima facie contradictory observations, by showing that domain antiregistration typically occurs as a metastable state, but can be kinetically preferred. The role of kinetics in governing registration/antiregistration is explored, the theory's predictions confirmed and illustrated with simulations, which show how a bilayer in the ``spinodal region'' can require nucleation to equilibrate. Our results shed light on a novel statistical mechanical problem of great practical importance, and motivate future work on intra- and inter-leaflet behaviour of bilayers. Reference: JJW and PDO, arXiv:1408.2744.   

X-ray Scattering Experiments Support Tilt-dependent Membrane Theory

Recent molecular dynamics simulations have suggested that the traditional model for topographical fluctuations in lipid bilayers should be enriched to consider molecular tilt. We present the first experimental support for a tilt-dependent theory. X-ray scattering from a liquid crystalline stack of oriented fluid phase lipid bilayers was collected and compared to the predictions of tilt-dependent and tilt-independent membrane models. Both models satisfactorily fit the X-ray data dominated by in-plane lengths greater than membrane thickness ($>$ 100~\AA), but only the tilt-dependent model accounts for X-ray data primarily attributable to shorter length correlations. By fitting the measured X-ray scattering intensity, both the bending modulus $K_c$ = 8.3 $\pm$ 0.6 $\times$ $10^{-20}$ J and the tilt modulus $K_\theta$ = 95 $\pm$ 7 mN/m were determined for DOPC bilayers at 30 $^\circ$C. Our experimental results support the enrichment of the classic Helfrich continuum model to include an internal degree of freedom, the fluctuations of lipid directors from the local normal.   

Two-Point Microrheology of Phase Separated Domains in Lipid Bilayers

Though the importance of membrane fluidity for cellular function has been well established for decades, methods for measuring lipid bilayer viscosity remain challenging to devise and implement. Recently, approaches based on characterizing the Brownian dynamics of individual tracers such as colloidal particles or lipid domains have provided insights into bilayer viscosity. In general, however, methods based on single-particle trajectories can be biased by distortions induced by the tracers, and furthermore provide a limited view of hydrodynamic response. The technique of two-point microrheology, in which correlations between the Brownian dynamics of pairs of tracers report on the properties of the intervening medium, resolves these issues, but has never been applied to lipid systems. We present the first two-point microrheological study of lipid bilayers, examining the correlated motion of domains in phase-separated lipid vesicles and comparing one- and two-point results. We measure correlation functions in excellent agreement with the forms predicted by two-dimensional hydrodynamic models, which reveal a viscosity that corresponds to the average of the lipid phases rather than the viscosity of the local neighborhood of the tracer.   

Use of Impedance Spectroscopy to Probe Changes in Mitochondrial Membrane Potential

The synthesis of ATP is driven by proton gradient and electrical potential across the mitochondrial inner membrane. It's electrical properties correlates with its physiological and pathological status. Electrical impedance spectroscopy is a non-invasive and relatively low cost technique where the impedance measurements monitors the underlying biological processes to determine parameters for biomarker studies. In this work we implement a multi frequency ($1kHz-10MHz$) bio electrical impedance to describe the changes in the electrical properties of mitochondria. The experimental strategy involved treating isolated mitochondria with the substrate succinate ($200mM$) in vivo to stimulate the activity of succinate dehydrogenase. Subsequent variability is introduced by the addition of different trifluorocarbonylcyanide-phenylhydrazone ($25\mu M < [FCCP] < 50 \mu M$) and dopamine ($50nM < [DA] < 0.5M$) concentration. We observe that succinate alone lead to an increase in the mitochondrial membrane potential $\Delta\Psi_m$. Dielectric spectroscopy measurements show a direct correlation between FCCP concentration and impedance and higher DA concentrations display marked decrease of membrane potential indicating a significantly reduced mitochondrial respiratory control.   

Proton-Pumping Mechanism in Complex I of Mitochondria Membrane

Mitochondria are the powerhouses of animal cells and also many bacteria. Complex I is the first enzyme in the mitochondrial respiratory chain, the process leading to storage of energy in the form of Adenosine Triphosphate (ATP). The structure of the enzyme was recently resolved and its functionality was correlated to the motion of a helical protein structure. However, the actual mechanism of the electron assisted proton-pumping of Complex I has remained mysterious because the electron (e-) and proton (H$+)$ pathways are well separated by a distance of up to 15 nm making the direct interaction of these charges negligible. We model the helix assisted indirect coupling between the electron and proton pathways as a non-uniformly charged piston oscillating between the coupled sites of a 3 site series system. The energy conversion is determined by single e- and H$+$ transport events. The piston oscillates between that central proton and electron sites and modulates their energy, while the coupling with other sites is weak and negligible. We show that with realistic values of parameters, this structure allows for proton pumping against the potential gradient.   

Thinning silicon-based membranes with electron irradiation for solid-state nanopore sensors

We present a controlled electron irradiation-based method to thin free-standing amorphous silicon membranes to less than 2 nm. Thinning is carried out in a scanning transmission electron microscope using a 200 keV electron probe to sputter silicon atoms. The transmitted electrons, scattered elastically and inelastically, are used as feedback signals to monitor and control the thinning process with sub-nanometer precision. Solid-state nanopore single-molecule sensors were fabricated by drilling a nanopore in the thinned membranes with the electron probe. These sensors operate in aqueous electrolyte and register passage of individual molecules by measuring changes in ionic conductance. We show that these solid-state nanopore single-molecule sensors sustain changes in ionic conductance with signal-to-noise ratios close to 100 at 100 kHz for translocations of double-stranded DNA in 1 M KCl electrolyte at room temperature coupled with conductance blockade of 60-95{\%}.   

Nanoscopic modulated phases in dDPPC:DLPC membranes studied with small angle neutron scattering

The lipid raft hypothesis states that functional, small (20-200nm) lateral heterogeneities in the cell membrane arise from the preferential association of proteins, sphingolipids, and cholesterol. Studies of model systems in the past decade have shown the formation of two liquid phases, a liquid-ordered and the typical fluid or liquid-disordered phase in ternary mixtures of a saturated lipid, unsaturated lipid, and cholesterol. These model raft systems on both a nanoscopic and macroscopic level have exhibited circular domains, but these are not the only possible shapes of phase separated domains. Giant vesicles extracted from live cells studied with fluorescence microscopy can exhibit critical behavior, showing distinctly fluctuating and not circular domains. Non-circular domains have also been observed in quarternary component GUVs in the form of modulated or patterned phases. We used small angle neutron scattering (SANS) to study the nanoscopic phase behavior of the well-studied lipid mixture DPPC:DLPC as a function of temperature. We applied an existing ab initio program to reconstruct the membrane without a priori shape fixing. Modulated phases are shown to persist at the nanoscale in small vesicles.   

Heterogeneous Rotational Diffusion in Lipid Monolayers

We have developed a new time-resolved fluorescence platform which enables us to follow the molecular orientation and dynamics of a lipid monolayer at the air - water interface. The rotational correlation time of the lipid probe NBD-PC is measured using fluorescence anisotropy for two lipid species. We measure the rotational diffusion in a monolayer of DPPC which displays a phase transition at room temperature from the liquid-expanded to the liquid-condensed phase. The constant rotational diffusion of the probe throughout the phase transition reflects the measurement of dynamics in only the liquid-expanded phase. We contrast the dynamic changes during this phase coexistence to the continuous density increase observed in DMPC at room temperature. We observe a non-exponential decay of the probe diffusion consistent with heterogeneity of the orientational dynamics; as the free-volume is reduced the diffusion becomes increasingly heterogeneous.   

Scaling and Alpha-helix Regulation of Protein Relaxation in a Lipid Bilayer

Protein conformation and orientation in the lipid membrane play a key role in many cellular processes. Here we use molecular dynamics simulation to investigate the relaxation and C-terminus diffusion of a model helical peptide: beta-amyloid (A$\beta )$ in a lipid membrane. We observed that after the helical peptide was initially half-embedded in the extracellular leaflet of phosphatidylcholine (PC) or PC/cholesterol (PC/CHOL) membrane, the C-terminus diffused across the membrane and anchored to PC headgroups of the cytofacial lipid leaflet. In some cases, the membrane insertion domain of the A$\beta $ was observed to partially unfold. Applying a sigmoidal fit to the process, we found that the characteristic velocity of the C-terminus, as it moved to its anchor site, scaled with $\theta _{\mathrm{u}}^{-4/3}$, where $\theta_{\mathrm{u}}$ is the fraction of the original helix that was lost during a helix to coil transition. Comparing this scaling with that of bead-spring models of polymer relaxation suggests that the C-terminus velocity is highly regulated by the peptide helical content, but that it is independent of the amino acid type. The A$\beta $ was stabilized by the attachment of the positive Lys28 side chain to the negative phosphate of PC or 3$\beta $ oxygen of CHOL in the extracellular lipid leaflet and of the C-terminus to its anchor site in the cytofacial lipid leaflet.   

Redistribution of Cholesterol by Membrane Active Peptides Alamethicin and Melittin

Many membrane active peptides are found to disrupt lipid bilayer of membrane in a concentration-dependent manner and form transmembrane pore over threshold concentrations, as depicted in Two-State Model. However, at low concentration, the interaction between peptide and lipid bilayer remains less understood beyond the thinning effect. Here we present small-angle neutron scattering studies of the interaction of two well-known membrane active peptides (melittin and alamethicin) with lipid bilayers made of dymyristoyl phosphatidylcholine (DMPC) and cholesterol (Chol). Through the use of deuterium-labeled DMPC, changes in the distribution of the lipid and cholesterol in unilamellar vesicles were observed for peptide concentrations well below those that drive pore formation. We have found the binding of the peptides have profound impact on the distribution of cholesterol residing inside the lipid bilayer. Those results point the existence of a possible secondary mechanism of action against cellular membranes as metabolic inhibitors that affect cellular machinery by redistributing cholesterol.   

Energetics of interactions between protein inclusions in lipid bilayer membranes

Proteins that are situated in cell membranes play vital roles in numerous cell functions. Examples include the enabling of the selective passage of ions or molecules into and out of cells, or the relaying of signals between cell interiors and their external environments. We develop a framework for understanding the energetics of the interactions that can arise between protein inclusions. We focus on the setting of inclusions in lipid bilayers and interactions that stem from the spatial overlap of the distortions induced by the inclusions in the orientational organization of the lipid molecules that constitute the bilayers.