Session X40: Proteins in Membranes

Copper coordination in the Glycine receptor by electron spin resonance

We describe the use of Electron Spin Resonance (ESR) to identify the coordination environment of copper in the extracellular domain of the protein, as well as the number of copper atoms that bind to Glycine receptor (GlyR). The GlyR channel mediates inhibitory neurotransmission in the central nervous system. It belongs to the superfamily of nicotincoid receptors. These receptors are formed by pentameric arrangement of subunits, each sharing a common topology having a large extracellular domain (ECD) and a transmembrane (TM) domain comprised of four membrane-spanning segments (TM1-TM4). For GlyR, four subunits (1-4) and one subunit have been identified to date, although the homomeric expression of just the $\alpha $1 subunit of GlyR is sufficient to reconstitute native-like activity. The results are expected to shed light on the role of metals ion in modulating ion permeation in such receptor. In addition, an identification of copper binding sites will allow the measurement of large range distance constraints in the receptor by pulsed ESR. Such structural information on the GlyR in various allosteric states is essential in order to shed light on the gating mechanism of this protein membrane.   

Probing interaction of antimicrobial peptide duramycin with lipid monolayers

Antimicrobial peptides are group of peptides which disrupt the microbial cell membrane through hydrophobic insertion into the outer lipid layer. Duramycin is a small tetracyclic peptide antibiotic, which has recently been shown to bind specifically to phosphatidylethanolamine (PE) lipids. We report the interaction of duramycin with phospholipid monolayers at air-water interface, studied using vibrational sum-frequency generation spectroscopy (VSFG) and fluorescence microscopy (FM). For monolayers containing PE lipids, VSFG reveals binding of duramycin to the monolayer through the appearance of a vibrational peak at 3045 cm$^{-1}$, corresponding to the C-H stretching vibration of phenylalanine amino acid. In addition, the amide I vibrational region shows that peptide has a $\beta $-sheet conformation. Similar experiments performed on phosphatidylcholine (PC) monolayers show the interaction is specific with PE.   

Alamethicin Structure in Lipid Bilayers

This investigation uses x-ray diffuse scattering and MD simulations to study alamethicin (Alm) in fully hydrated bilayers of DOPC and diC22:1PC. Comparison of the experimental and simulated form factors supports the standard conclusion that Alm helices are inserted transmembrane along the bilayer normal at high humidity and high concentrations. Little change in membrane thickness with inserted Alm helices occurs for DOPC up to 1/10 Alm/DOPC. By contrast, the x-ray data strongly indicate that the diC22:1PC membrane, which is thicker than DOPC by 7 {\AA}, thins with added Alm. Fitting the data to models of the electron density gives a decrease in thickness of 4 {\AA} at 1/10 Alm/diC22:1PC. Although Alm's helical length is close to the hydrophobic thickness of DOPC (27 {\AA}), it is mismatched with the thicker diC22:1PC. Alm decreases the bending modulus (K$_{C})$ by a factor of $\sim $2 in DOPC and a factor $\sim $10 in diC22:1PC membranes at P/L $\sim $1/10. Determination of the B modulus reveals a large increase in Hamaker parameter when Alm is added to diC22:1PC, but not to DOPC.   

Alamethicin Supramolecular Organization in Membranes

In this work we investigate the effect of membrane hydration and hydrophobic mismatch on the Alm channel superstructure in an oriented multilayer sample by x-ray scattering. Wide angle x-ray (WAXS) scattering near 14 nm$^{-1}$ indicates that the lipid chain region is not perturbed much by the incorporation of up to 10 mole percent Alm. Low angle x-ray scattering (LAXS) indicates that when the sample is very dry, which promotes interactions between neighboring bilayers, a body centered tetragonal crystal packing of Alm channels is formed. As the hydration level increases closer to biological conditions, the separation between bilayers increases, the interbilayer interactions weaken, and the crystalline order disappears while considerable diffuse scattering remains. The effect of hydrophobic mismatch is examined for two mono-unsaturated lipids, diC18:1PC and diC22:1PC, that differ in bilayer thickness by 0.7nm. There is also in-plane scattering at a medium q of 7nm$^{-1}$ that our analysis suggests may not be from the Alm channel structure.   

Geometric cue for protein localization in a bacterium

Proteins in bacteria deploy to particular places, but the cues for localization are frequently mysterious. We present evidence that the membrane protein SpoVM recognizes a geometric cue in \textit{Bacillus subtilis}. In vivo experiments show that SpoVM localizes to a particular patch of the inner membrane in sporulating \textit{Bacillus subtilis} bacteria, namely the convex surface of the developing spore. Our in vitro experiments support the hypothesis that this localization is driven by geometry rather than biochemical recognition. When purified SpoVM is incubated with polydisperse micrometer-sized DOPC vesicles, we observe that the protein preferentially adsorbs on smaller vesicles, of diameter similar to the size of the bacterial spore ($\sim$ 1 $\mu$m). Using fluorescent GFP-tagged SpoVM, we quantify the amount of adsorbed protein by confocal microscopy. Our results, when interpreted using existing protein adsorption models, suggest the existence of a cooperative adsorption mechanism for high enough membrane curvature, which involves the formation of small clusters of proteins. Membrane curvature could be a general cue for protein localization in bacteria.   

The effect of protein on phase separation in giant unilamellar lipid vesicles.

We explore the coarsening and out of plane curvature (budding) of domains in lipid bilayer vesicles composed of DOPC (unsaturated), PSM (saturated), and cholesterol. Green fluorescent protein (GFP) was added to the membrane in controlled amounts by binding to the Ni-chelating lipid, Ni-DOGS. Vesicles with diameters between 10 and 50 microns were prepared via a standard electroformation procedure. As a sample is lowered through temperature Tmix, a previously homogeneous vesicle phase separates into two fluid phases with distinct compositions. Phase-separated domains have a line tension (energy/length) at the boundary with the major phase which competes with bending energy and lateral tension to determine the overall configuration of the vesicle. Domain budding and coarsening were observed and recorded using both bright field and fluorescence microscopy during temperature scans and with varying concentrations of GFP. The addition of a model protein into our system allows for a broader understanding of the effect of protein, which are ubiquitous in cell membranes, on phase separation, budding, and coarsening.   

Kinetics and Thermodynamics of Peptide (pHLIP) insertion and folding in a lipid bilayer

We study spontaneous insertion and folding across a lipid bilayer of moderately polar membrane peptide pHLIP - pH Low Insertion Peptide. pHLIP has three major states: soluble in water or bound to the surface of a lipid bilayer as an unstructured monomer, and inserted across the bilayer as a monomeric $\alpha $-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to calculate the transition energies between states. The free energy of binding to a surface of lipid bilayer is about -7 kcal/mol and the free energy of insertion and folding across a lipid bilayer at low pH is nearly -2 kcal/mol. We performed stopped-flow fluorescence and CD measurements to elucidate molecular mechanism of pHLIP insertion and folding within a lipid bilayer and to calculate the activation energy of formation of transmembrane helix. pHLIP also has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. We plan to discuss a number of related kinetics and thermodynamic parameters from our measurements.   

Effects of Proteins and Lipids on Each Other in a Simulated Non-equilibrium Biomembrane Model

Lateral organization in biomembranes plays a major role in membrane topology, and is thus implicated in many basic functions of biomembranes such as endocytosis and signal transduction. In this study, non-equilibrium Monte Carlo simulations are used to investigate two related scenarios: 1. the effect of a rigid distribution of proteins on the lateral organization of lipids in a biomembrane, and 2. the degree to which lipid interactions influence the lateral organization of membrane-associated proteins that are free to translate laterally. Our model includes generic saturated and unsaturated lipids, proteins, and cholesterol, and is driven out of equilibrium through simulated endo- and exo-cytosis events. By varying the temperature, the protein mole fraction, and the interaction strengths, we examine the conditions under which various types of lateral organization occur. Simulation results are analyzed with pair-correlation functions and the Ripley K-test. We compare results from simulations of the two scenarios above and from simulations of biomembranes lacking protein.   

Interaction of Defensins with Model Cell Membranes

Antimicrobial peptides (AMPs) comprise a key component of innate immunity for a wide range of multicellular organisms. For many AMPs, activity comes from their ability to selectively disrupt and lyse bacterial cell membranes. There are a number of proposed models for this action, but the detailed molecular mechanism of selective membrane permeation remains unclear. Theta defensins are circularized peptides with a high degree of selectivity. We investigate the interaction of model bacterial and eukaryotic cell membranes with theta defensins RTD-1, BTD-7, and compare them to protegrin PG-1, a prototypical AMP, using synchrotron small angle x-ray scattering (SAXS). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane phase behavior induced by these different peptides we will discuss the importance of amino acid composition and placement on membrane rearrangement.   

Membrane fluidity in the presence of membrane-binding peptides

Arf proteins are GTP-ases that participate in vesicle trafficking inside cells. They are able to interact with membranes through their N-terminus when they are bound to GTP, and they detach from the membrane when GTP is hydrolyzed. The N-terminus of Arf1 (amino acids 2-17) folds into an amphipathic helix that can insert into lipid bilayers. Arf1 is also myristoylated; it has myristic acid, a 14-carbon fatty acid `tail', attached to it. We set out to test the hypothesis that the binding of the myristoylated N-terminus of Arf1 to lipid membranes changes the mechanical properties of the membrane, in ways that myristic acid alone or amphipathic peptides alone do not. We use three reporter molecules embedded in vesicles, whose fluorescence emission spectrum depends on the properties of the environment in which they are found, to measure three distinct aspects of membrane fluidity: Bispyrene is sensitive to lateral motion along the membrane, Prodan's emission gives a measure of the packing of the head groups, and DPH polarization reflects the packing of the hydrophobic tails. We will present effects found for four molecules (myristic acid, myristoylated and non-myristoylated N-terminus of Arf1, and the ALPS domain of KES) in a concentration-dependent manner, and discuss the importance of these results in the vesicle-trafficking picture.   

New insights into the bacterial cell wall peptidoglycan architecture

The molecular architecture of the bacterial cell wall peptidoglycan (sacculi) is among the most challenging, yet still unsolved, structural problems in biochemistry. Two models have been proposed: the planar model, in which the glycan strands lie in the plane of the cell surface, and the scaffold model, in which the glycan strands lie perpendicular to the cell surface. We have used atomic force microscopy (AFM) to investigate the molecular structure of this unique biopolymer in the rod-shaped bacterium Bacillus subtilis at high resolution. AFM images recorded in air on single sacculi revealed a porous regular network with 25-50 nm-wide peptidoglycan fibers and a 5-25 nm pore size. Interestingly, the new bacterial pole showed a regular structure with the same fiber sizes but with the fibers running in a direction that is almost perpendicular to that observed away from the pole. This finding combined with our previous data on live hydrated bacteria (1) provides new insights into the three-dimensional architecture of the peptidoglycan of Gram-positive bacteria. 1- A. Touhami, M. H. Jericho, and T. J. Beveridge, J. Bacteriol., 2004 186: 3286-3295.   

An Analytic Study of Molecular Motion in Cell Membranes

We present a theoretical calculation to describe the confined motion of transmembrane molecules in cell membranes. Understanding the motion of membrane-associated molecules, e.g. various types of receptors, has great modern relevance in cell biology. Our study is divided into two parts. In the first, we consider motion in an ordered system and in the second, we investigate the effects of disorder by employing an effective medium approximation. Both are based on Master equations for the probability of the molecules moving as random walkers, and leads to explicit usable solutions including expressions for the molecular mean square displacement and effective diffusion constants. As a result, the calculations make possible, in principle, the extraction of confinement parameters such as mean compartment sizes and mean intercompartmental transition rates from experimentally reported published observations.   

Detergent interaction with tethered bilayer lipid membranes for protein reconstitution

Tethered bilayer lipid membranes (tBLMs) are self-assembled biomimetic structures in which the membrane is separated from a solid substrate by a nm-thick hydrated submembrane space. These model systems are being used in binding studies of peripheral proteins and exotoxins. Here we aim at their application for the reconstitution of water-insoluble integral membrane proteins. As an alternative to fusion of preformed proteoliposomes we study the direct reconstitution of such proteins for applications in biosensing and pharmaceutical screening. For reconstitution, highly insulating tBLMs ($R\sim10^{5}-10^{6} ~\Omega$) were temporarily incubated with a detergent to screen for conditions that keep the detergent-saturated membranestable and ready to incorporate detergent-solubilized proteins. We assess the electrical characteristics, i.e. specific resistance and capacitance, by means of electrochemical impedance spectroscopy (EIS) under timed incubation with decylmaltoside and dodecylmaltoside detergents in a regime around their critical micelle concentration, 1.8 mM and 0.17 mM respectively and demonstrate the restoration of the tBLM upon detergent removal. Thereby a range of concentration and incubation times was identified, that represents optimal conditions for the subsequent membrane protein reconstitution.