Session U17: Proteins in Membranes and the Cytoskeleton

Hierarchy of Specific Lipid-Peptide Interactions Produces the Activity of Cell-penetrating and Cell-permeating Peptides

Protein transduction domains can cross cell membranes with high efficiency, even when carrying a variety of cargos, and thus has strong biotechnological potential. The molecular mechanism of entry, however, is not well understood. We use small-angle x-ray scattering (SAXS) and confocal microscopy to systematically study the interaction of the TAT and ANTP PTD with model membranes of variable composition. Their membrane transduction activity requires the presence of both PE and PS lipids in the membrane. Antimicrobial peptides (AMP's) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogs of AMP's, such as the family of phenylene ethynylene antimicrobial oligomers (AMO's), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. PE lipid greatly enhances permeating activity of AMO in these membranes, showing the importance of specific lipid composition for the activity of cell-permeating peptides. Since bacterial cell membranes are richer in PE lipids than are eukaryotic cell membranes, this may indicate a mechanism for antimicrobial specificity.   

Simulations of the pore structures for a M2G1yR derived channel forming peptide in membrane

In an effort to develop a peptide-based compound suitable for clinical use as a channel replacement therapeutic for treating channelopathies such as cystic fibrosis, we present a reductionist model that appears to capture many of the biophysical properties of an intact ion channel using short channel-forming peptides. We have developed two anion selective channel-forming peptides with near native and altered properties from the peptides derived from the glycine receptor: NK$_{4}$-M2GlyR-p22 WT (KKKKPAR-VGLGITTVLTMTTQS) and NK$_{4}$-M2GlyR-p22 S22W (KKKKPARVGLGITTVLTMTTQW), respectively. Starting with the two structures determined by solution multidimensional NMR (800 MHz) in SDS, we used CHARMM and NAMD to perform molecular dynamics simulations on the monomers. Using the existing experimental data, we then built an initial 5- helix assembly by altering the tilted angle, rotational angle and pore radius. We investigated the impact of the single mutation at position 22 on the structure and dynamics of the pore formed in a membrane build in a hydrated POPC lipid bilayer. Probable structures for both assemblies are presented.   

Membrane-mediated mechanism of amyloid oligomer toxicity in Alzheimer's Disease.

There is strong evidence, that soluble amyloid $\beta $ (A$\beta )$ oligomers, involved in Alzheimer's Disease, are the primary toxic species of A$\beta $, although the mechanism of cell toxicity is very much debated [1]. Neutron reflectivity and electrical impedance spectroscopy assess the structural impact of A$\beta $ (1-42) oligomers and their effect on the electrical properties of a tethered phosphocholine model membrane. Two distinct and reversible peptide -- membrane interactions were revealed: At low A$\beta $ concentrations an equal incorporation of A$\beta $ into both lipid leaflets and a compaction of the lipid membrane takes place. A$\beta $ locally lowers the dielectric barrier for ion transport and the activation energy for ion transport through the bilayer remains significantly above that of a water-filled transmembrane pore. At high A$\beta $ concentrations, an additional membrane thinning is observed. [1] D. Eliezer, J. Gen. Physiol. 128:631 (2006).   

Molecular target of synthetic antimicrobial oligomer in bacterial membranes

Antimicrobial peptides comprises a key component of innate immunity for a wide range of multicellular organisms. It has been shown that natural antimicrobial peptides and their synthetic analogs have demonstrated broad-spectrum antimicrobial activity via permeating bacterial membranes selectively. Synthetic antimicrobials with tunable structure and toxicological profiles are ideal for investigations of selectivity mechanisms. We investigate interactions and self-assembly using a prototypical family of antimicrobials based on phenylene ethynylene. Results from synchrotron small angle x-ray scattering (SAXS) results and in vitro microbicidal assays on genetically modified `knock-out' bacteria will be presented.   

Interaction of Arginine-Rich Peptides with Model Cell Membranes

Cell-penetrating peptides have the ability to traverse the plasma membrane of eukaryotic cells. Furthermore, these peptides can transport cargo across a range of cell membranes, implying they have many potential biotechnological applications. In this study we compare the interaction of three commonly used arginine-rich cell-penetrating peptides, TAT, Penetratin, and pVEC, with model cell membranes of variable charge density and intrinsic curvature, using synchrotron small angle x-ray scattering (SAXS). To better understand the respective roles of arginine and hydrophobic residues in membrane reorganization we also examine the interaction of arginine-leucine (R60L20) block copolypeptides with model membranes, as well as the relationship between membrane composition and peptide induced changes in membrane topology.   

Lipid and Protein Sorting during Membrane Tube Formation

Motivated by recent experiments that implicate the mechanical properties of membranes in lipid sorting, we examine the interplay of lipid composition and curvature in membrane tubes. We study how the dependence of bending stiffness and surface tension on membrane lipid and protein composition affects tube formation. Drawing a tube from a vesicle leads to a rearrangement of composition in which the phase of higher flexibility segregates in the tube, the region of high mean curvature. For point forcing, the force vs. extension curve can have a sharp drop just as the tube begins to form.   

Equilibrium Stability of Transmembrane Proteins : A Hard-Core Gas Problem.

Hydropathy plots, a moving average of amino acid hydrophobicity over a sequence, can be used to predict potential protein structure, in particular transmembrane proteins. Traditionally transmembrane regions are identified by peaks above an empirical cutoff. We treat the transmembrane segments as a one-dimensional gas of hard rods in a correlated random energy landscape. At zero temperature, where the entropic contribution due to the loops is negligible, we calculate the density profile as a function of the chemical potential in the case of the original as well as randomly generated landscapes. The density profile exhibits plateaus indicating regions where a transmembrane segment has been established. For designed versus random sequences we explore the distribution of the sizes of these plateaus and attempt to infer characteristic features that may be interpreted in terms of the stability of the protein in its inserted state.   

Diffusion of Transmembrane Proteins: Beyond the Saffman-Delbr\"{u}ck Model

The hydrodynamic model of Saffmann and Delbr\"{u}ck [\textit{PNAS} \textbf{72} 3111 (1975)] predicts that the diffusion constant $D$ of proteins embedded in a fluid membrane exhibits a weak logarithmic dependence on the radius $a$ of the protein. However, recent experiments by Gambin \textit{et al.} [\textit{PNAS} \textbf{103} 2098 (2006)] have observed a much stronger 1/$a$ dependence for proteins embedded in model membranes. Local interactions between a transmembrane protein and the lipids that surround it can cause the lipids to deform by, for example, stretching or compressing their tails, or by tilting their long axis with respect to the membrane's surface. In this talk, we show that these deformations lead to additional sources of energy dissipation which cause the protein diffusion constant \textit{D $\sim $ 1/a}, as observed by Gambin \textit{et al.} Our model incorporates the lipid stretch and tilt degrees of freedom into a traditional hydrodynamic model by introducing additional scalar and vector fields, respectively.   

Lateral organization of a non-equilibrium membrane model with immobile randomly-distributed impurities

Cell membranes are dynamic, far-from-equilibrium systems; transport, signaling, and other membrane functions ensure that membrane lateral organization is heterogeneous across several length scales. However, many studies and simulations consider membranes as equilibrium systems. Here, we present a model of cell membranes that includes simplified endo- and exocytosis and immobile randomly-distributed impurities. The impurities take the form of fixed protein sites within the membrane, which act as potential localization zones for micro-domain rafts. We analyze the lateral organization in terms of spatial statistics through a modified Ripley K-test. This model illuminates the role of protein in a 1:1:1 mixture of saturated lipids, unsaturated lipids, and cholesterol. Additionally, this model exhibits a realistic heterogeneity of cluster sizes and shapes, and suggests conditions under which we may observe a partitioning of cholesterol in the membrane. Such simulated observations of the direct interactions between cholesterol, lipids, and protein on the molecular scale can enhance our understanding of all biophysical processes occurring within cell membranes.   

Multidrug efflux transporter activity in sea urchin embryos:Does localization provide a diffusive advantage?

Experiments have shown upregulation of multidrug efflux transporter activity approximately 30 min after fertilization in the sea urchin embryo [1]. These ATP-hydrolyzing transporter proteins pump moderately hydrophobic molecules out of the cell and represent the cell's first line of defense againstexogenous toxins. It has also been shown that transporters are moved in vesicles along microfilaments and localized to tips of microvilli prior to activation. We have constructed a geometrically realistic model of the embryo, including microvilli, to explore the functional role of this localization in the efficient elimination of toxins from the standpoint of diffusion. We compute diffusion of toxins in extracellular, membrane and intracellular spaces coupled with transporter activity, using experimentally derived values for physical parameters. For transporters uniformly distributed along microvilli and tip-localized transporters we compare regions in parameter space where each distribution provides diffusive advantage, and comment on the physically expected conditions. [1] A. M. Hamdoun, G. N. Cherr, T. A. Roepke and D. Epel, Developmental Biology {\bf 276} 452 (2004).   

Observation of a single particle diffusive motion on the membrane by 3-dimensional particle tracking microscope.

Three-dimensional motion of particles on the surface of giant vesicle membranes has been tracked with nanometer scale resolution using a quadrant photodiode, which is used as a position sensitive detector (PSD), placed in the diffraction pattern formed behind a 100x microscopic objective. Two He-Ne beams were focused to trap a single particle in solution between cover slips with 100 micro-meter gap. The single particle is brought very close to the surface of a membrane system by optical tweezer. By reducing the optical power, the particle sits on the membrane surface and moves diffusively. The diffusing motion is measured using a fast-feedback controller designed to respond to the axial and the lateral position of the particle simultaneously in less than 250 micro-seconds. This work has better positional and temporal accuracy of 3-dimensional particle tracking than conventional video-tracking methods.   

Oxygen dynamics in photosynthetic membranes.

Production of oxygen by oxygenic photosynthetic organisms is expected to raise oxygen concentration within their photosynthetic membranes above normal aerobic values. These raised levels of oxygen may affect function of many proteins within photosynthetic cells. However, experiments on proteins \textit{in vitro }are usually performed in aerobic (or anaerobic) conditions since the oxygen content of a membrane is not known. Using theory of diffusion and measured oxygen production rates we estimated the excess levels of oxygen in functioning photosynthetic cells. We show that for an individual photosynthetic cell suspended in water oxygen level is essentially the same as that for a non-photosynthetic sell. These data suggest that oxygen protection mechanisms may have evolved after the development of oxygenic photosynthesis in primitive bacteria and was driven by the overall rise of oxygen concentration in the atmosphere. Substantially higher levels of oxygen are estimated to occur in closely packed colonies of photosynthetic bacteria and in green leafs.   

Identification of co-evolving sites in the ligand binding domain of G protein-coupled receptors using mutual information

G protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins in humans. They are involved in signal transduction in numerous cellular processes and are the most common target for pharmacological intervention via activation or inhibition. Identification of functionally important sites is relevant for better understanding the ligand-receptor interaction and therefore for drug delivery. In a superfamily of proteins, functionally important but co-evolving sites are not easily identified in a multiple sequence alignment (MSA). Using a MSA of trans-membrane (TM) domains of GPCR superfamily, we identify sites which co-evolve, and may therefore be functionally important. Assigning the TM site as a node and the MI of site pairs as an inverse inter-node distance, a MI graph is established. Co-evolving sites are then identified via this graph. Nodes characterized by high connectivity are located within the commonly accepted ligand binding site of GPCRs, suggesting that concerted co-evolution of a number of neighboring residues gave rise to a multitude of subfamilies each recognizing a specific set of ligands. MI and graph analysis may serve as a tool for the identification of topologically conserved binding pockets in the families of evolutionarily related proteins.   

Dynamics of Actin Cable Polymerization in Fission Yeast

In fission yeast, formin for3p nucleates actin filament bundles (cables) at cell tips which contribute to polarized cell growth. Actin cables reach a steady state of dynamic turnover involving for3p-mediated actin polymerization at the barbed ends near the plasma membrane, retrograde flow of polymerized actin toward the cell center, and cable disassembly. Formin for3p associates with actin at the cable tip where it transiently polymerizes actin filaments and subsequently follows the retrograde actin cable flow (Martin and Chang, Curr. Biol. 16, 1161, 2006). Because of the small number of formin nucleators, the actin cable dynamics are subject to spatial and temporal fluctuations. We studied actin cable dynamics with simple analytical models and whole cell computational models which combine deterministic simulation of actin diffusion with stochastic simulation of formin reaction and diffusion. Our model successfully explains a large number of experimental observations, such as density of formin speckles and variance of actin cable density. The model predicts significant spatial gradient of actin and formin molecules in the cytoplasm, powered by the retrograde flow of actin cables.   

Synchrotron X-ray Diffraction Study of Neurofilament Networks Interaction under Osmotic Pressure

Neurofilaments (NFs) are cytoskeletal proteins, which are found abundantly in nerve cell axons and impart mechanical stability and act as structural scaffolds for microtubules. The filaments assemble from 3 different subunit proteins to form a 10 nm diameter flexible polymers with radiating unstructured sidearms. At high protein concentration, the NFs form a nematic hydrogel network with a well-defined interfilament spacing as measured by synchrotron small angle x-ray scattering (SAXS) [1]. Here, NFs purified from bovine spinal cord are reassembled in vitro. Using analogous SAXS-osmotic pressure techniques [2] we study forces between NFs and directly probe the polyampholyte brush interactions between NF sidearms. We measure the interfilament spacing at different osmotic pressure, salt and sidearm concentrations. The study reveals the non-trivial electrostatic nature of the interfilament interaction within the NF hydrogel. [1] J. Jones, C.R. Safinya (submitted) [2] D. J. Needleman et al., PRL 93, 198104 (2004)