Polymer Nanodiscs and Their Bioanalytical Potential

Membrane proteins (MPs) play a pivotal role in cellular function and are therefore predominant pharmaceutical targets. Although detailed understanding of MP structure and mechanistic activity is invaluable for rational drug design, challenges are associated with the purification and study of MPs. This review delves into the historical developments that became the prelude to currently available membrane mimetic technologies before shining a spotlight on polymer nanodiscs. These are soluble nanosized particles capable of encompassing MPs embedded in a phospholipid ring. The expanding range of reported amphipathic polymer nanodisc materials is presented and discussed in terms of their tolerance to different solution conditions and their nanodisc properties. Finally, the analytical scope of polymer nanodiscs is considered in both the demonstration of basic nanodisc parameters as well as in the elucidation of structures, lipid–protein interactions, and the functional mechanisms of reconstituted membrane proteins. The final emphasis is given to the unique benefits and applications demonstrated for native nanodiscs accessed through a detergent free process.     

Combination of the CHARMM27 force field with united-atom lipid force fields

Computer simulations offer a valuable way to study membrane systems, from simple lipid bilayers to large transmembrane protein complexes and lipid-nucleic acid complexes for drug delivery. Their accuracy depends on the quality of the force field parameters used to describe the components of a particular system. We have implemented the widely used CHARMM22 and CHARMM27 force fields in the GROMACS simulation package to (i) combine the CHARMM22 protein force field with two sets of united-atom lipids parameters; (ii) allow comparisons of the lipid CHARMM27 force field with other lipid force fields or lipid-protein force field combinations. Our tests do not show any particular issue with the combination of the all-atom CHARMM22 force field with united-atoms lipid parameters, although pertinent experimental data are lacking to assess the quality of the lipid-protein interactions. The conversion utilities allow automatic generation of GROMACS simulation files with CHARMM nucleic acids and protein parameters and topologies, starting from pdb files using the standard GROMACS pdb2gmx method. CMAP is currently not implemented.     

The Role of Amyloid β-Biomembrane Interactions in the Pathogenesis of Alzheimer's Disease: Insights from Liposomes as Membrane Models

The aggregation and deposition of amyloid β (Aβ) peptide onto neuronal cells, with consequent cellular membrane perturbation, are central to the pathogenesis of Alzheimer's disease (AD). Substantial evidence reveals that biological membranes play a key role in this process. Thus, elucidating the mechanisms by which Aβ interacts with biomembranes and becomes neurotoxic is fundamental to developing effective therapies for this devastating progressive disease. However, the structural basis behind such interactions is not fully understood, largely due to the complexity of natural membranes. In this context, lipid biomembrane models provide a simplified way to mimic the characteristics and composition of membranes. Aβ-biomembrane interactions have been extensively investigated applying artificial membrane models to elucidate the molecular mechanisms underlying the AD pathogenesis. This review summarizes the latest findings on this field using liposomes as biomembrane model, as they are considered the most promising 3D model. The current challenges and future directions are discussed.     

A kinetic model for the effects of vanadate on human erythrocyte membrane

The effects of vanadate on human erythrocyte membrane have been investigated with stopped-flow and equilibrium fluorescence quenching techniques. The equilibrium study showed a half-quenching concentration (K_(1/2)) of 0.27 mmol·L~(-1). The stopped-flow experiment exhibited a fast rise (t_(1、2)~f～1s) and a slow drop (t_(1/2)~s 1～2 min) in fluorescence. Based on the results and that from the across membrane transport of vanadate, a kinetic model is proposed which suggests that the membrane proteins experience a series of conformational changes before and during the quenching of the intrinsic fluorescence. These changes are induced mainly by three kinds of interactions: (i) the long-distance, non-specific interaction between the vanadate and the erythrocyte membrane surface, (ⅱ) the charge interaction between the vanadate and parts of the membrane proteins, and(ⅲ) the binding of the vanadate to some membrane proteins.     

Formation of pH‐Resistant Monodispersed Polymer–Lipid Nanodiscs

Polymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near‐native environment. Despite the recent advances in the development and usage of polymer lipid nanodisc systems, lack of control over size and poor tolerance to pH and divalent metal ions are major limitations for further applications. A facile modification of a low‐molecular‐weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra‐stability towards divalent metal ion concentration over a pH range of 2.5 to 10. The macro‐nanodiscs (>20 nm diameter) show magnetic alignment properties that can be exploited for high‐resolution structural studies of membrane proteins and amyloid proteins using solid‐state NMR techniques. The new polymer, SMA‐QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.     

Template-Free Self-Assembly of Artificial De Novo Viral Coat Proteins into Nanorods: Effects of Sequence,Concentration, and Temperature

The self-assembly of protein polymers is a promising route to prepare sophisticated functional nanostructures. However, the interplay between protein self-assembly by itself and its co-assembly with a template is not well understood. Silk-based protein polymers that co-assemble with DNA to form rod-like artificial viruses are herein developed and the effects of silk block length, concentration, and temperature in the self-assembly of the proteins alone are characterized by using a combination of bulk dynamic light scattering (DLS) and single-molecule atomic force microscopy (AFM). Protein nanorods were slowly formed (up to hours) through the interaction of the silk-like blocks. The proteins present a silk-length dependent critical elongation concentration, and above it the amount and size of nanorods rapidly increase. Temperature-dependent light scattering data was adequately fitted into a cooperative model of nucleation–elongation. These results are also important to understand the self-assembly of designed viral coat proteins with DNA templates to form artificial virus-like particles and help us to define general guidelines to design proteins with the ability to precisely organize matter at the nanoscale.     

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s⁻¹ was determined for residues protected from exchange. Transfer within the protein, as tracked in the ¹⁵N-¹H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s⁻¹ for several resolved residues, explaining the site specificity.     

脂双层膜表面结构与稳定性的原子力显微镜研究

用原子力显微镜研究了1,2－二油酸甘油－3－磷酸－1甘油（DOPG）脂双层膜 的表面结构与稳定性。实验结果表明,原子力显微镜的探针与脂双层膜的相互作用 导致脂双层膜表面产生一个永久的损伤。静电相互作用对脂双层膜结构和稳定性的 影响表明,在NaCl溶液中制成的脂质体,随着NaCl浓度的增加,它们的双层膜更稳 定。在低的NaCl浓度则经常被损伤,在1 mol/L NaCl溶液中制备的指双层变得更稳 定。在KCl溶液中结果恰好相反。在高的KCl浓度中经常被损伤,随着KCl浓度的降 低,它们的双层膜更稳定。葡萄糖和蔗糖对脂双层膜结构有稳定作用。     

基于仿生膜的功能化单纳米通道在分析化学中的应用

灵感来源于蛋白质离子通道的仿生功能化单纳米通道,已逐渐成为一种成熟的单分子检测技术和离子整流器。功能化纳米通道包括两种:基因改造的蛋白质纳米通道和固体加工的纳米通道。常用的固体纳米通道有三种:在纳米氮化硅或石墨烯上用聚焦离子束(FIB)或电子束(FEB)轰击得到的纳米通道,化学腐蚀聚合物薄膜中的重金属离子轨迹得到的锥形纳米通道和拉制毛细管或激光刻蚀得到的玻璃纳米孔。相对于蛋白质纳米通道,固态的人工纳米通道具有更优越的机械稳定性,并可用于各种功能基团的修饰。经过近十年的发展,包括蛋白质纳米通道在内的各种仿生的纳米通道已广泛用于对小分子、蛋白质和聚合物等其他一些对象的定性和定量检测。本综述详细介绍了近年来国内外该领域的发展,并对未来的发展方向作了简要的展望。     

Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.     

Anisotropic Self‐Assembly of Citrate‐Coated Gold Nanoparticles on Fluidic Liposomes

The behavior of self‐assembly processes of nanoscale particles on plasma membranes can reveal mechanisms of important biofunctions and/or intractable diseases. Self‐assembly of citrate‐coated gold nanoparticles (cAuNPs) on liposomes was investigated. The adsorbed cAuNPs were initially fixed on the liposome surfaces and did not self‐assemble below the phospholipid phase transition temperature (T_m). In contrast, anisotropic cAuNP self‐assembly was observed upon heating of the composite above the T_m, where the phospholipids became fluid. The number of self‐assembled NPs is conveniently controlled by the initial mixing ratio of cAuNPs and liposomes. Gold nanoparticle protecting agents strongly affected the self‐assembly process on the fluidic membrane.     

Monolayer and Bilayer Formation of Molecular 2D Networks Assembled at the Liquid/Solid Interfaces by Solution-Based Drop-Cast Method

In recent years, extending self-assembled structures from two-dimensions (2D) to three-dimensions (3D) has been a paradigm in surface supramolecular chemistry and contemporary nanotechnology. Using organic molecules of p-terphenyl-3,5,3′,5′-tetracarboxylic acid (TPTC), and scanning tunneling microscopy (STM), we present a simple route, that is the control of the solute solubility in a sample solution, to achieve the vertical growth of supramolecular self-assemblies, which would otherwise form monolayers at the organic solvent/graphite interface. Presumably, the bilayer formations were based on π-conjugated overlapped molecular dimers that worked as nuclei to induce the yielding of the second layer. We also tested other molecules, including trimesic acid (TMA) and 1,3,5-tris(4-carboxyphenyl)-benzene (BTB), as well as the further application of our methodology, demonstrating the facile preparation of layered assemblies.     

DNA-Mediated Self-Assembly and Metallization of Semiconductor Nanorods for the Fabrication of Nanoelectronic Interfaces

DNA nanostructures provide a powerful platform for the programmable assembly of nanomaterials. Here, this approach is extended to semiconductor nanorods that possess interesting electrical properties and could be utilized for the bottom-up fabrication of nanoelectronic building blocks. The assembly scheme is based on an efficient DNA functionalization of the nanorods. A complete coverage of the rod surface with DNA ensures a high colloidal stability while maintaining the rod size and shape. It furthermore supports the assembly of the nanorods at defined docking positions of a DNA origami platform with binding efficiencies of up to 90 % as well as the formation of nanorod dimers with defined relative orientations. By incorporating orthogonal binding sites for gold nanoparticles, defined metal-semiconductor heterostructures can be fabricated. Subsequent application of a seeded growth procedure onto the gold nanoparticles (AuNPs) allows for to establish a direct metal-semiconductor interface as a crucial basis for the integration of semiconductors in self-assembled nanoelectronic devices.     

Aromatic Fluorination of Multiblock Amphiphile Enhances Its Incorporation into Lipid Bilayer Membranes

We designed multiblock amphiphiles AmF and AmH , which consist of perfluorinated and non-fluorinated hydrophobic units, respectively. Absorption spectroscopy revealed that both amphiphiles are molecularly dispersed in organic solvent, while they form aggregates under aqueous conditions. Furthermore, we investigated whether AmF and AmH can be incorporated into DOPC lipid bilayer membranes, and found that the maximum concentration of AmF that can be incorporated into DOPC lipid bilayer membranes is 43 times higher than that of AmH .     

Effect of different treatments of long-range interactions and sampling conditions in molecular dynamic simulations of rhodopsin embedded in a dipalmitoyl phosphatidylcholine bilayer

The present study analyzes the effect of the simulation conditions on the results of molecular dynamics simulations of G-protein coupled receptors (GPCRs) performed with an explicit lipid bilayer. Accordingly, the present work reports the analysis of different simulations of bovine rhodopsin embedded in a dipalmitoyl phosphatidylcholine (DPPC) lipid bilayer using two different sampling conditions and two different approaches for the treatment of long-range electrostatic interactions. Specifically, sampling was carried out either by using the statistical ensembles NVT or NPT (constant number of atoms, a pressure of 1 atm in all directions and fixed temperature), and the electrostatic interactions were treated either by using a twin-cutoff, or the particle mesh Ewald summation method (PME). The results of the present study suggest that the use of the NPT ensemble in combination with the PME method provide more realistic simulations. The use of NPT during the equilibration avoids the need of an a priori estimation of the box dimensions, giving the correct area per lipid. However, once the system is equilibrated, the simulations are irrespective of the sampling conditions used. The use of an electrostatic cutoff induces artifacts on both lipid thickness and the ion distribution, but has no direct effect on the protein and water molecules.     

Solubilization of Membrane Proteins into Functional Lipid‐Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer

Once removed from their natural environment, membrane proteins depend on membrane‐mimetic systems to retain their native structures and functions. To this end, lipid‐bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane‐protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl‐chain order, does not interfere with optical spectroscopy in the far‐UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations.     

Model Biological Membranes and Possibilities of Application of Electrochemical Impedance Spectroscopy for their Characterization

Biological membranes are essential parts of living systems. They represent an interface between intracellular and extracellular space. Depending on their structure, they often perform very complex functions and play an important role in the transport of both charged and uncharged particles in any organism. Structure of the biological membranes, which play very important role in electrochemical processes inside living organisms, is very complicated and still not precisely defined and explained. Model lipid membranes are used to gain detail information about properties of real biological membranes and about associated electrochemical processes. Electrochemistry, especially electrochemical impedance spectroscopy (EIS), can play a useful role in the characterization of properties of model lipid membranes (planar and supported lipid bilayers, tethered lipid membranes, liposomes, etc.). This review is focused on model biological membranes and the possibilities and limitations of electrochemical methods and namely of EIS in this field.     

双层类脂膜修饰玻碳电极研究去甲肾上腺素电化学行为及其分析应用

报道了去甲肾上腺素在双层类脂膜修饰玻碳电极上的电化学行为。在 0 .8～ - 0 .3V(vs.Ag/AgCl)电位范围内 ,于 pH 6 .5、0 .0 1mol/LKH2 PO4 Na2 HPO4底液中 ,去甲肾上腺素产生很灵敏的氧化峰电流。该电流与去甲肾上腺素浓度在 3.5× 10 -6～ 3.1× 10 -4mol/L范围内呈良好线性关系。该电极可作为检测去甲肾上腺素的新型的高灵敏度电化学生物传感器。     

Performance of the general amber force field in modeling aqueous POPC membrane bilayers

The aim of this work was to answer the question of whether the general amber force field (GAFF) is good enough to simulate fully hydrated POPC membrane bilayers. The test system contained 128 POPC and 2985 TIP3P water molecules. The equilibration was carried out in a nonarbitrary manner to reach the stable liquid-crystalline phase. The simulations were performed by using particle mesh Ewald electrostatics implemented in molecular dynamics packages Amber8 (NPT ensembles) and NAMD2 (NPgammaT ensembles). The computational results were assessed against the following experimental membrane properties: (i) area per lipid, (ii) area compressibility modulus, (iii) order parameter, (iv) gauche conformations per acyl chain, (v) lateral diffusion coefficients, (vi) electron density profile, and (vii) bound water at the lipid/water interface. The analyses revealed that the tested force field combination approximates the experimental values at an unexpectedly good level when the NPgammaT ensemble is applied with a surface tension of 60 mN m(-1) per bilayer. It is concluded that the GAFF/TIP3P combination can be utilized for aqueous membrane bilayer simulations, as it provides acceptable accuracy for biomolecular modeling.