Session A41: Focus Session: Structure and Dynamics of Membranes

A Neutron View of Proteins in Lipid Bilayers

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly-charged S1-S4 voltage- sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated potassium channels. We have used neutron diffraction, solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations, cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings reveal that voltage sensors have evolved to interact with the lipid membrane while keeping the energetic and structural perturbations to a minimum, and that water penetrates into the membrane to hydrate charged residues and shape the transmembrane electric field.   

Curvature Forces in Membrane Lipid-Protein Interactions

Membrane protein conformational changes, folding, and stability may all involve elastic deformation of the bilayer. Non-specific properties of the bilayer play a significant role in modulating protein conformational energetics. A flexible-surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. The FSM describes elastic coupling of membrane lipids to integral membrane proteins. Curvature and hydrophobic matching to the lipid bilayer entails a stress field that explains membrane protein stability. Rhodopsin provides an important example, where solid-state NMR and FTIR spectroscopy characterize the energy landscape of the dynamically activated receptor. Time-resolved UV-visible and FTIR spectroscopic studies show how membrane lipids affect the metarhodopsin equilibrium due to non-specific material properties. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress on rhodopsin function are all explained by the new biomembrane model. By contrast, the older fluid-mosaic model fails to account for such effects on membrane protein activity. According to the FSM proteins are regulated by membrane lipids whose spontaneous curvature most closely matches the activated state within the lipid membrane.   

Thickness fluctuations in pure lipid bilayers

Recently neutron spin echo (NSE) revealed dynamical processes in a surfactant membrane system around the length scale of the membrane thickness where the classical Helfrich treatment breaks down. This excess dynamics (on top of the bending fluctuations) observed in a nonionic surfactant, water and oil system, was attributed to thickness fluctuations of the membrane. In the case of bilayers formed with the nonionic surfactant in water the thickness fluctuation amplitude was estimated to be a few angstroms. In the study presented here, we apply the technique to explore thickness fluctuations in lipid bilayers. The result shows clear evidence of thickness fluctuations above $T_{m}$, where the lipid tails display liquid ordering, while none are discernable below $T_{m}$. The estimated amplitude of the observed membrane thickness fluctuations is approximately 4 {\AA}. These results are consistent with theoretical expectation and recent molecular dynamics simulations. Varying the lipid tail length from 14 carbons to 18 carbons per tail does not appear to affect to the dynamics very much.   

A Simple Analysis of Small-Angle Neutron Scattering Data to Estimate Thickness Fluctuations of the Membrane

Small-angle neutron scattering (SANS) and neutron spin echo (NSE) experiments are one of the most important laboratory techniques to investigate structure and dynamic properties of biological and nanotechnology-related membrane systems. Due to the sensitivity of about 1~100 nm length scales, these experimental techniques provide extensive information over a wide variety of technological and scientific applications. Recently, the author and his colleagues studied swollen lamellar structure systems consisting of nonionic surfactant, water, and oil using SANS/NSE and molecular dynamics (MD) simulation. They proposed a new experimental technique to measure the thickness fluctuations of surfactant layers and verified their approach using MD simulations. In this talk a possible simpler approach to estimate the membrane thicknesses and fluctuations directly from the isotropic scattering intensities in the two-dimensional SANS profile will be proposed. Generally, this characteristic feature is reproduced using various scattering theories to estimate the membrane thickness; however, the thickness fluctuation amplitude has never been estimated from the SANS profile. The results obtained from the approach will be compared with the experimental results obtained by Nagao and co-workers.   

High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with {\AA}ngstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P$_{3}$, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P$_{2})$ act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P$_{2}$. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration $<$ 5 {\AA}) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains. In the bound state, PTEN's regulatory C-terminal tail is displaced from the membrane and organized on the far side of the protein, $\sim $~60 {\AA} away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. Molecular dynamics simulations, currently in progress, refine this structural picture further.   

Application of Small-Angle Neutron and X-ray Scattering in Determining Lipid Bilayer Structure

Accurately determining lipid structure in biologically relevant fluid bilayers is not straightforward. We have recently developed a hybrid experimental/computational technique (i.e., the scattering density profile, or SDP model), which exploits the fact that neutron and X-ray scattering are sensitive to different bilayer thicknesses - the large difference in neutron scattering length density (SLD) between proteated lipid and deuterated water defines the overall bilayer thickness, while X-ray scattering resolves the headgroup-headgroup distance due to the large scattering contrast between the electron-rich phosphate groups and the hydrocarbon/aqueous medium. A key step in the SDP analysis is the use of MD simulations to parse the lipid molecule into fragments whose volume probability distributions follow simple analytical functional forms. Given the appropriate atomic scattering lengths, these volume probabilities can simultaneously predict both the neutron and X-ray SLD profiles, and hence the scattering form factors. Structural results for commonly used phosphatidylcholine and phosphatidylglycerol lipids will be given.   

Growth Mechanism of Lipid-Based Nanodiscs -- a Model Membrane for Studying Kinetics of Particle Coalescence

Lipid-based nanodiscs composed of long- and short- chain lipids [namely, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG) and dihexanoyl phosphatidylcholine (DHPC)] constantly form at high lipid concentrations and at low temperatures (i.e., below the melting transition temperature of DMPC, T$_{M})$. The initial size of these nanodiscs (at high total lipid concentration, C$_{L}>$ 20 wt.{\%}) is relatively uniform and of similar dimension (according to dynamic light scattering and small angle neutron scattering experiments), seemingly independent of thermal history. Upon dilution, the nanodiscs slowly coalesce and grow in size with time irreversibly. Our preliminary result shows that the growth rate strongly depends on several parameters such as charge density, C$_{L}$ and temperature. We have also found that the nanodisc coalescence is a reaction limit instead of diffusion limit process through a time-resolved study.   

Swellable Model POPC/POPG/DHPC Membrane with a Lamellar Long-Range Order

A physiological relevant biomimetic model membrane is of great necessity for the structural characterization of membrane protein. This presentation will report a small-angle neutron scattering (SANS) result on two lipid bicellar series composed of 1-palmitoyl-2-oleoyl-\textit{sn}-glycero-3-phosphocholine(POPC)/1,2-dihexanoyl-\textit{sn}-glycero-3-phosphocholine (DHPC) and POPC/DHPC/1-palmitoyl-2-oleoyl-\textit{sn}-glycero-3-phospho-(1'-\textit{rac}-glycerol) (POPG). Instead of the multi-lamellae vesicle (MLV) structure observed in zwitterionic POPC/DHPC mixture, the perforated lamellae (PL) structure is found in POPC/POPG/DHPC upon addition of small amount of charged lipid, POPG {\{}R=[POPG]/([POPC]+[POPG])=0.01{\}}. The PL phase exists from 10 to 60 degree C and the interlamellar spacing (d-spacing) varies from 12.9 to 49.0 nm as the lipid concentration changes from 25 to 7.5{\%} wt where the lamellae still indicate long-range order. The effect of temperature and charge density (R) on structural variation will be discussed in this presentation.   

Mobility of water and selected atoms in bilayer DMPC membranes

Molecular dynamics simulations have been used to determine the mobility of water molecules as a function of their positions in a fully hydrated free standing DMPC membrane at 303 K. In a 10 \AA{} thick water layer with bulk density just outside the membrane, the mobility of the water molecules is reduced by about a factor of two relative to bulk. For water molecules penetrating deeper into the membrane there is an increasing reduction in the mobility with up to two orders of magnitude for those deepest into the membrane. A comparison with the mobility of selected atoms in the lipid molecules shows that about 5 water molecules/lipid molecule move on the same time scale as the lipid molecules and may therefore be considered to be so tightly bound to them that they essentially follow their motion. The simulation results are quantitatively compared with quasielastic neutron scattering results on single-supported bilayer DMPC membranes.   

Structure of single-supported DMPC lipid bilayer membranes as a function of hydration level studied by neutron reflectivity and Atomic Force Microscopy

We have recently been investigating the diffusion of water on single-supported DMPC lipid bilayer membranes at different levels of hydration, using high-resolution quasielastic neutron scattering (QNS). To aid in the interpretation of these QNS studies, we have conducted neutron reflectivity (NR) measurements on SPEAR at LANSCE to characterize the structure of similarly prepared samples. Protonated DMPC membranes were deposited onto SiO$_{2}$-coated Si(100) substrates and characterized by Atomic Force Microscopy (AFM) at different levels of hydration. We find reasonable agreement between the membrane thickness determined by NR and AFM at room temperature. We also find consistency between the scattering length density (SLD) profile in the vicinity of the upper leaflet of the supported DMPC membrane and that found in a molecular dynamics simulation of a freestanding membrane at 303 K. However, the fit to the reflectivity curve can be improved by modifying the SLD profile near the leaflet closest to the SiO$_{2}$ surface.   

Cholesterol Flip-Flop Dynamics in a Phospholipid Bilayer: A 10 Microsecond All-Atom Molecular Dynamics Simulation

Cholesterol (CHOL) molecules play a key role in modulating the rigidity of cell membranes, and controlling intracellular transport and signal transduction. Using all-atom molecular dynamics and the parallel replica approach, we study the effect of CHOL molecules on mechanical stresses across a dipalmitoylphosphatidycholine (DPPC)-CHOL bilayer, and the mechanism by which CHOL molecules migrate from one bilayer leaflet to the other (flip-flop events). On average, we observe a CHOL flip-flop event in half-a-microsecond. Once a CHOL flip-flop event is triggered, the inter-leaflet migration occurs in about 62 nanoseconds. The energy barrier associated with flip-flop events is found to be 73 kJ/mol. Results for membrane rigidity as a function of CHOL concentration will also be presented.