Frank Heinrich

Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes

Surface-tethered biomimetic bilayer membranes (tethered bilayer lipid membranes (tBLMs)) were formed on gold surfaces from phospholipids and a synthetic 1-thiahexa(ethylene oxide) lipid, WC14. They were characterized using electrochemical impedance spectroscopy, neutron reflection (NR), and Fourier-transform infrared reflection-absorption spectroscopy (FT-IRRAS) to obtain functional and structural information. The authors found that electrically insulating membranes (conductance and capacitance as low as 1 μS cm−2 and 0.6 μF cm−2, respectively) with high surface coverage (>95% completion of the outer leaflet) can be formed from a range of lipids in a simple two-step process that consists of the formation of a self-assembled monolayer (SAM) and bilayer completion by “rapid solvent exchange.” NR provided a molecularly resolved characterization of the interface architecture and, in particular, the constitution of the space between the tBLM and the solid support. In tBLMs based on SAMs of pure WC14, the hexa(ethylene oxide) tether region had low hydration even though FT-IRRAS showed that this region is structurally disordered. However, on mixed SAMs made from the coadsorption of WC14 with a short-chain “backfiller,” ß-mercaptoethanol, the submembrane spaces between the tBLM and the substrates contained up to 60% exchangeable solvent by volume, as judged from NR and contrast variation of the solvent. Complete and stable “sparsely tethered” BLMs (stBLMs) can be readily prepared from SAMs chemisorbed from solutions with low WC14 proportions. Phospholipids with unsaturated or saturated, straight or branched chains all formed qualitatively similar stBLMs.

Structure of functional Staphylococcus aureus α-hemolysin channels in tethered bilayer lipid membranes

We demonstrate a method for simultaneous structure and function determination of integral membrane proteins. Electrical impedance spectroscopy shows that Staphylococcus aureus alpha-hemolysin channels in membranes tethered to gold have the same properties as those formed in free-standing bilayer lipid membranes. Neutron reflectometry provides high-resolution structural information on the interaction between the channel and the disordered membrane, validating predictions based on the channels x-ray crystal structure. The robust nature of the membrane enabled the precise localization of the protein within 1.1 A. The channels extramembranous cap domain affects the lipid headgroup region and the alkyl chains in the outer membrane leaflet and significantly dehydrates the headgroups. The results suggest that this technique could be used to elucidate molecular details of the association of other proteins with membranes and may provide structural information on domain organization and stimuli-responsive reorganization for transmembrane proteins in membrane mimics.

HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid.

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.

Structure and properties of tethered bilayer lipid membranes with unsaturated anchor molecules.

The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [Z-20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol] and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection-absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients relative to those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore-forming toxin α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicate that HC18 results in tBLMs with improved physical properties for the incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution are imperative.

An ion-channel-containing model membrane: structural determination by magnetic contrast neutron reflectometry

To many biophysical characterisation techniques, biological membranes appear as two-dimensional structures with details of their third dimension hidden within a 5 nm profile. Probing this structure requires methods able to discriminate multiple layers a few Ångströms thick. Given sufficient resolution, neutron methods can provide the required discrimination between different biochemical components, especially when selective deuteration is employed. We have used state-of-the-art neutron reflection methods, with resolution enhancement via magnetic contrast variation to study an oriented model membrane system. The model is based on the Escherichia coli outer membrane protein OmpF fixed to a gold surface via an engineered cysteine residue. Below the gold is buried a magnetic metal layer which, in a magnetic field, displays different scattering strengths to spin-up and spin-down neutrons. This provides two independent datasets from a single biological sample. Simultaneous fitting of the two datasets significantly refines the resulting model. A β-mercaptoethanol (βME) passivating surface, applied to the gold to prevent protein denaturation, is resolved for the first time as an 8.2 ± 0.6 Å thick layer, demonstrating the improved resolution and confirming that this layer remains after OmpF assembly. The thiolipid monolayer (35.3 ± 0.5 Å), assembled around the OmpF is determined and finally a fluid DMPC layer is added (total lipid thickness 58.7 ± 0.9 Å). The dimensions of trimeric OmpF in isolation (53.6 ± 2.5 Å), after assembly of lipid monolayer (57.5 ± 0.9 Å) and lipid bilayer (58.7 ± 0.9 Å), are precisely determined and show little variation.

Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions.

Zooming in on disordered systems: Neutron reflection studies of proteins associated with fluid membranes

Neutron reflectometry (NR) is an emerging experimental technique for the structural characterization of proteins interacting with fluid bilayer membranes under conditions that mimic closely the cellular environment. Thus, cellular processes can be emulated in artificial systems and their molecular basis studied by adding cellular components one at a time in a well-controlled environment while the resulting structures, or structural changes in response to external cues, are monitored with neutron reflection. In recent years, sample environments, data collection strategies and data analysis were continuously refined. The combination of these improvements increases the information which can be obtained from NR to an extent that enables structural characterization of protein-membrane complexes at a length scale that exceeds the resolution of the measurement by far. Ultimately, the combination of NR with molecular dynamics (MD) simulations can be used to cross-validate the results of the two techniques and provide atomic-scale structural models. This review discusses these developments in detail and demonstrates how they provide new windows into relevant biomedical problems. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Solid supported lipid membranes: New concepts for the biomimetic functionalization of solid surfaces

Surface-layer (S-layer( supported lipid membranes on solid substrates are interfacial architectures mimicking the supramolecular principle of cell envelopes which have been optimized for billions of years of evolution in most extreme habitats. The authors implement this biological construction principle in a variety of layered supramolecular architectures consisting of a stabilizing protein monolayer and a functional phospholipid bilayer for the design and development of new types of solid-supported biomimetic membranes with a considerably extended stability and lifetime — compared to existing platforms — as required for novel types of bioanalytical sensors. First, Langmuir monolayers of lipids at the water/air interface are used as test beds for the characterization of different types of molecules which all interact with the lipid layers in various ways and, hence, are relevant for the control of the structure, stability, and function of supported membranes. As an example, the interaction of S-layer proteins from the bulk phase with a monolayer of a phospholipid synthetically conjugated with a secondary cell wall polymer (SCWP) was studied as a function of the packing density of the lipids in the monolayer. Furthermore, SCWPs were used as a new molecular construction element. The exploitation of a specific lectin-type bond between the N-terminal part of selected S-layer proteins and a variety of glycans allowed for the buildup of supramolecular assemblies and thus functional membranes with a further increased stability. Next, S-layer proteins were self-assembled and characterized by the surface-sensitive techniques, surface plasmon resonance spectroscopy and quartz crystal microbalance with dissipation monitoring. The substrates were either planar gold or silicon dioxide sensor surfaces. The assembly of S-layer proteins from solution to solid substrates could nicely be followed in-situ and in real time. As a next step toward S-layer supported bilayer membranes, the authors characterized various architectures based on lipid molecules that were modified by a flexible spacer separating the amphiphiles from the anchor group that allows for a covalent coupling of the lipid to a solid support, e.g., using thiols for Au substrates. Impedance spectroscopy confirmed the excellent charge barrier properties of these constructs with a high electrical resistance. Structural details of various types of these tethered bimolecular lipid membranes were studied by using neutron reflectometry. Finally, first attempts are reported to develop a code based on a SPICE network analysis program which is suitable for the quantitative analysis of the transient and steady-state currents passing through these membranes upon the application of a potential gradient.

An Accurate In Vitro Model of the E. coli Envelope.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir–Blodgett and Langmuir–Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.

Continuous distribution model for the investigation of complex molecular architectures near interfaces with scattering techniques.

Biological membranes are composed of a thermally disordered lipid matrix and therefore require non-crystallographic scattering approaches for structural characterization with x-rays or neutrons. Here we develop a continuous distribution (CD) model to refine neutron or x-ray reflectivity data from complex architectures of organic molecules. The new model is a flexible implementation of the composition-space refinement of interfacial structures to constrain the resulting scattering length density profiles. We show this model increases the precision with which molecular components may be localized within a sample, with a minimal use of free model parameters. We validate the new model by parameterizing all-atom molecular dynamics (MD) simulations of bilayers and by evaluating the neutron reflectivity of a phospholipid bilayer physisorbed to a solid support. The determination of the structural arrangement of a sparsely-tethered bilayer lipid membrane (stBLM) comprised of a multi-component phospholipid bilayer anchored to a gold substrate by a thiolated oligo(ethylene oxide) linker is also demonstrated. From the model we extract the bilayer composition and density of tether points, information which was previously inaccessible for stBLM systems. The new modeling strategy has been implemented into the ga_refl reflectivity data evaluation suite, available through the National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR).