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.     

Reconstitution of the Cytb5–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full‐length membrane‐bound P450 and its redox partner cytochrome b₅ (cytb₅) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane‐bound complex in model membranes renders high‐resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450–cytb₅ complex in peptide‐based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450–cytb₅ complex in the nanodisc. This is the first successful demonstration of a protein–protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane‐bound protein complexes.     

Nanoassembly of Oligopeptides and DNA Mimics the Sequential Disassembly of a Spherical Virus

Protein subunits of a low aspect ratio (width over length) with stimuli‐responsiveness are hallmark building blocks of spherical viruses. The interaction of these repeating subunits enables hierarchical assembly for genome packaging and sequential disassembly for optimal genome release. Here, we mimicked these features and constructed a functional spherical artificial virus. The rationally designed 22‐amino acid peptide containing pH‐sensitive histidines and aromatic residues self‐assembled into homogenous nanodiscs of a low aspect ratio (≈7×7×4 nm). In the presence of DNA, the inter‐nanodisc interactions drove the formation of a viral capsid‐like structure enclosing DNA in the interior. This artificial virus roughly 50 nm in diameter underwent partial disassembly in response to acidic pH. The resulting intermediate with lowered DNA‐binding affinity continued to protect DNA from nuclease digestion. Such nanostructures, which mimic the intricate morphology and the intracellular transformation of spherical viruses, can be useful for constructing synthetic gene delivery vehicles, as evidenced by their efficient transgene expression.     

CHARMM-GUI Nanodisc Builder for modeling and simulation of various nanodisc systems

Nanodiscs are discoidal protein–lipid complexes that have wide applications in membrane protein studies. Modeling and simulation of nanodiscs are challenging due to the absence of structures of many membrane scaffold proteins (MSPs) that wrap around the membrane bilayer. We have developed CHARMM-GUI Nanodisc Builder ( http://www.charmm-gui.org/input/nanodisc ) to facilitate the setup of nanodisc simulation systems by modeling the MSPs with defined size and known structural features. A total of 11 different nanodiscs with a diameter from 80 to 180 Å are made available in both the all-atom CHARMM and two coarse-grained (PACE and Martini) force fields. The usage of the Nanodisc Builder is demonstrated with various simulation systems. The structures and dynamics of proteins and lipids in these systems were analyzed, showing similar behaviors to those from previous all-atom and coarse-grained nanodisc simulations. We expect the Nanodisc Builder to be a convenient and reliable tool for modeling and simulation of nanodisc systems. © 2019 Wiley Periodicals, Inc.     

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.     

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K⁺ channel proteins are assembled through zinc‐finger protein (ZFP)‐adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP‐fused Kir3 channels and ZFP‐based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K⁺ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.     

DNA-Mediated Stack Formation of Nanodiscs

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.     

Vesicle Tubulation with Self‐Assembling DNA Nanosprings

A major goal of nanotechnology and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane‐deforming proteins, we designed DNA‐origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA‐coated membrane tubules emerge from spherical vesicles when DNA‐origami polymerization or high membrane‐surface coverage occurs. Unlike many previous methods, the DNA self‐assembly‐mediated membrane tubulation eliminates the need for detergents or top‐down manipulation. The DNA‐origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphology, underscoring the intricate interplay between lipid bilayers and vesicle‐deforming DNA structures.     

Aligning nanodiscs at the air-water interface, a neutron reflectivity study

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 ? with a surface coverage of 66 ± 4%. This layer is located about 15 ? below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 ?) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.     

Inhibition of the In Vitro Replication of DNA by an Aptamer–Protein Complex in an Autonomous DNA Machine

DNA replication plays a central role in living organisms. Unregulated or uncontrollable DNA replication is well known to result in many pathological states, such as cancer, autoimmune diseases, and viral/bacterial infections. We report that an aptamer–protein complex could indirectly inhibit in vitro replication of DNA. An isothermal DNA machine based on the strand‐displacement amplification is employed to support our assumption. An antithrombin aptamer sequence is rationally encoded into the DNA replication template. Once thrombin binds to the template, the as‐formed aptamer–protein complexes can, in turn, become a barrier to the polymerase and inhibit the DNA replication activities in both static and dynamic modes. The inhibition is successfully confirmed by both fluorescence and gel‐electrophoresis experiments. Considering the availability of a broad library of aptamers and the existence of various DNA/protein interactions, our results imply the possibility for the rational regulation of DNA replication in vivo.     

Interfacing Synthetic DNA Logic Operations with Protein Outputs

DNA logic gates are devices composed entirely of DNA that perform Boolean logic operations on one or more oligonucleotide inputs. Typical outputs of DNA logic gates are oligonucleotides or fluorescent signals. Direct activation of protein function has not been engineered as an output of a DNA‐based computational circuit. Explicit control of protein activation enables the immediate triggering of enzyme function and could yield DNA computation outputs that are otherwise difficult to generate. By using zinc‐finger proteins, AND, OR, and NOR logic gates were created that respond to short oligonucleotide inputs and lead to the activation or deactivation of a split‐luciferase enzyme. The gate designs are simple and modular, thus enabling integration with larger multigate circuits, and the modular structure gives flexibility in the choice of protein output. The gates were also modified with translator circuits to provide protein activation in response to microRNA inputs as potential cellular cancer markers.     

Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane Vesicles

We report a synthetic biology‐inspired approach for the engineering of amphipathic DNA origami structures as membrane‐scaffolding tools. The structures have a flat membrane‐binding interface decorated with cholesterol‐derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions leading to the formation of ordered arrays on the membrane. Such a tight and regular arrangement makes our DNA origami capable of deforming free‐standing lipid membranes, mimicking the biological activity of coat‐forming proteins, for example, from the I‐/F‐BAR family.     

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

The free solution electrophoretic behavior of DNA‐protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA‐protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA‐methyl‐CpG‐binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA‐protein complexes. This method is based on the effective separation of free DNA and DNA‐protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide‐coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA‐MBD2b complexes, we found that the values of (μ₀/μ)‐1 were directly proportional to the charge‐to‐mass ratios of formed complexes, where the μ₀ and μ are the mobility of free DNA probe and DNA‐protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge‐to‐mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA‐binding properties of the purified MBD2b protein and help to analyze other DNA‐protein complexes. Additionally, this study enhances the understanding of the influence of the charge‐to‐mass ratios of formed DNA‐protein complexes on their separation and electrophoretic behaviors.     

CHARMM‐GUI Martini Maker for modeling and simulation of complex bacterial membranes with lipopolysaccharides

A complex cell envelope, composed of a mixture of lipid types including lipopolysaccharides, protects bacteria from the external environment. Clearly, the proteins embedded within the various components of the cell envelope have an intricate relationship with their local environment. Therefore, to obtain meaningful results, molecular simulations need to mimic as far as possible this chemically heterogeneous system. However, setting up such systems for computational studies is far from trivial, and consequently the vast majority of simulations of outer membrane proteins still rely on oversimplified phospholipid membrane models. This work presents an update of CHARMM‐GUI Martini Maker for coarse‐grained modeling and simulation of complex bacterial membranes with lipopolysaccharides. The qualities of the outer membrane systems generated by Martini Maker are validated by simulating them in bilayer, vesicle, nanodisc, and micelle environments (with and without outer membrane proteins) using the Martini force field. We expect this new feature in Martini Maker to be a useful tool for modeling large, complicated bacterial outer membrane systems in a user‐friendly manner. © 2017 Wiley Periodicals, Inc.     

Charge Neutralization Drives the Shape Reconfiguration of DNA Nanotubes

Reconfiguration of membrane protein channels for gated transport is highly regulated under physiological conditions. However, a mechanistic understanding of such channels remains challenging owing to the difficulty in probing subtle gating‐associated structural changes. Herein, we show that charge neutralization can drive the shape reconfiguration of a biomimetic 6‐helix bundle DNA nanotube (6HB). Specifically, 6HB adopts a compact state when its charge is neutralized by Mg²⁺; whereas Na⁺ switches it to the expanded state, as revealed by MD simulations, small‐angle X‐ray scattering (SAXS), and FRET characterization. Furthermore, partial neutralization of the DNA backbone charges by chemical modification renders 6HB compact and insensitive to ions, suggesting an interplay between electrostatic and hydrophobic forces in the channels. This system provides a platform for understanding the structure–function relationship of biological channels and designing rules for the shape control of DNA nanostructures in biomedical applications.     

A Supercharged Fluorescent Protein as a Versatile Probe for Homogeneous DNA Detection and Methylation Analysis

Supercharged proteins are a new class of functional proteins with exceptional stability and potent ability to deliver bio‐macromolecules into cells. As a proof‐of‐principle, a novel application of supercharged proteins as a versatile biosensing platform for nucleic acid detection and epigenetics analysis is presented. Taking supercharged green fluorescent protein (ScGFP) as the signal reporter, a simple turn‐on homogenous method for DNA detection has been developed based on the polyionic nanoscale complex of ScGFP/DNA and toehold strand displacement. This assay shows high sensitivity and potent ability to detect single‐base mismatch. Furthermore, combined with bisulfite conversion, this ScGFP‐based assay was further applied to analyze site‐specific DNA methylation status of genomic DNA extracted from real human colon carcinoma tissue sample with ultrahigh sensitivity (4 amol methylated DNA).     

An Electropolymerized Membrane Biosensor for Speciffic DNA Recognition

A sensitive electrochemical biosensor for detecting the sequence of short DNA oligomers is represented. The biosensor is based on a platinum electrode covered a polymerized membrane of conductive monomer N‐[6‐(thien‐3‐yl)acetoxy]‐pyrrolidine‐2, 5‐dione (TAPD). The membrane of TAPD immobilizes a probe DNA on the electrode. The hybridization of the probe with a sequence‐specific DNA in sample solutions is monitored by a self‐synthesized electroactive indicator, which specifically intercalates in the hybrids on the electrode surface. The current signal of the biosensor is proportional to the concentration of the target DNA in samples, and a very low detection limit of 5 × 10^?10 mol/L is found. The biosensor has been used to detect the short oligomers containing of HTV‐1 and mycobacterrium nucleotide sequences.     

Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs

Nanodiscs have become a leading technology to solubilize membrane proteins for biophysical, enzymatic, and structural investigations. Nanodiscs are nanoscale, discoidal lipid bilayers surrounded by an amphipathic membrane scaffold protein (MSP) belt. A variety of analytical tools has been applied to membrane proteins in nanodiscs, including several recent mass spectrometry studies. Mass spectrometry of full-length proteins is an important technique for analyzing protein modifications, for structural studies, and for identification of proteins present in binding assays. However, traditional matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry methods for analyzing full-length membrane proteins solubilized in nanodiscs are limited by strong signal from the MSP belt and weak signal from the membrane protein inside the nanodisc. Herein, we show that an optimized ultra-thin layer MALDI sample preparation technique dramatically enhances the membrane protein signal and nearly completely eliminates the MSP signal. First-shot MALDI and MALDI imaging are used to characterize the spots formed by the ultra-thin layer method. Furthermore, the membrane protein enhancement and MSP suppression are shown to be independent of the type of membrane protein and are applicable to mixtures of membrane proteins in nanodiscs.     

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial‐scale systems aiming at plasmid DNA purification.