Reconstitution Of Membrane Proteins Research Articles

Formation of Giant Lipid Vesicle Containing Dual Functions Facilitates Outer Membrane Phospholipase.

Giant lipid vesicles are used to study artificial cell models, as well as the encapsulation of biomolecules, and the reconstitution of membrane proteins on these vesicles. Recently, complex reactions in giant vesicles have been controlled by reconstituting numerous kinds of biomolecules. However, it is challenging to generate giant lipid vesicles containing a diverse set of proteins at concentrations sufficient to ensure proper functioning. Here, we describe an artificial cell model showing dual functions of small molecule transportation and small vesicle budding, using a dual functional membrane protein (transportation and phosphatase activity) called the outer membrane phospholipase (OmpLA). To the best of our knowledge, we have revealed for the first time the transportation of ions or small molecules through OmpLA on the charged lipid bilayer. The lipid composition controlled the orientation of OmpLA through proteinase K digestion. Finally, OmpLA enzyme activity of phospholipid hydrolysis caused the budding of small vesicles.

Robust Photoelectric Biomolecular Switch at a Microcavity-Supported Lipid Bilayer.

Biomolecular devices based on photo-responsive proteins have been widely proposed for medical, electrical, and energy storage and production applications. Also, bacteriorhodopsin (bR) has been extensively applied in such prospective devices as a robust photo addressable proton pump. As it is a membrane protein, in principle, it should function most efficiently when reconstituted into a fully fluid lipid bilayer, but in many model membranes, lateral fluidity of the membrane and protein is sacrificed for electrochemical addressability because of the need for an electroactive surface. Here, we reported a biomolecular photoactive device based on light-activated proton pump, bR, reconstituted into highly fluidic microcavity-supported lipid bilayers (MSLBs) on functionalized gold and polydimethylsiloxane cavity array substrates. The integrity of reconstituted bR at the MSLBs along with the lipid bilayer formation was evaluated by fluorescence lifetime correlation spectroscopy, yielding a protein lateral diffusion coefficient that was dependent on the bR concentration and consistent with the Saffman–Delbrück model. The photoelectrical properties of bR-MSLBs were evaluated from the photocurrent signal generated by bR under continuous and transient light illumination. The optimal conditions for a self-sustaining photoelectrical switch were determined in terms of protein concentration, pH, and light switch frequency of activation. Overall, a significant increase in the transient current was observed for lipid bilayers containing approximately 0.3 mol % bR with a measured photo-current of 250 nA/cm2. These results demonstrate that the platforms provide an appropriate lipid environment to support the proton pump, enabling its efficient operation. The bR-reconstituted MSLB model serves both as a platform to study the protein in a highly addressable biomimetic environment and as a demonstration of reconstitution of seven-helix receptors into MSLBs, opening the prospect of reconstitution of related membrane proteins including G-protein-coupled receptors on these versatile biomimetic substrates.

Formation of Supported Lipid Bilayers Derived from Vesicles of Various Compositional Complexity on Conducting Polymer/Silica Substrates.

Supported lipid bilayers (SLBs) serve important roles as minimalistic models of cellular membranes in multiple diagnostic and pharmaceutical applications as well as in the strive to gain fundamental insights about their complex biological function. To further expand the utility of SLBs, there is a need to go beyond simple lipid compositions to thereby better mimic the complexity of native cell membranes, while simultaneously retaining their compatibility with a versatile range of analytical platforms. To meet this demand, we have in this work explored SLB formation on PEDOT:PSS/silica nanoparticle composite films and mesoporous silica films, both capable of transporting ions to an underlying conducting PEDOT:PSS film. The SLB formation process was evaluated by using the quartz crystal microbalance with dissipation (QCM-D) monitoring, total internal reflection fluorescence (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) for membranes made of pure synthetic lipids with or without the reconstituted membrane protein β-secretase 1 (BACE1) as well as cell-derived native lipid vesicles containing overexpressed BACE1. The mesoporous silica thin film was superior to the PEDOT:PSS/silica nanoparticle composite, providing successful formation of bilayers with high lateral mobility and low defect density even for the most complex native cell membranes.

Benchmarks of SMA-Copolymer Derivatives and Nanodisc Integrity.

Poly(styrene-co-maleic acid) or SMA and its derivatives, a family of synthetic amphipathic copolymers, are increasingly used to directly solubilize cell membranes to functionally reconstitute membrane proteins in native-like copolymer-lipid nanodiscs. Although these copolymers act, de facto, like a "macromolecular detergent", the polymer-based lipid-nanodiscs has been demonstrated to be an excellent membrane mimetic for structural and functional studies of membrane proteins and their complexes by a variety of biophysical and biochemical approaches. In many studies reported in the literature, the choice of the right SMA formulation can depend on a number of factors, and the experimental conditions are typically developed according to a trial-and-error process since each studied system requires adapted protocols. While increasing number of nanodisc-forming copolymers are reported to be useful and they provide flexibilities in optimizing the sample preparation conditions, it is important to develop a systematic protocol that can be used for various applications. In this context, there is a vital necessity of benchmarking the performances of existing copolymer formulations, assessing crucial parameters for the successful extraction, isolation, and stabilization of membrane proteins. In this study, we compare both copolymers and copolymer-lipid nanodiscs obtained by SMA-EA with a set of anionic XIRAN copolymer formulations commercially available under the names of SL25010 P, SL30010 P, and SL40005 P. The reported results show how the critical micellar concentration (c.m.c.) of each copolymer is significantly altered in the presence of lipids and confirms the existence of an equilibrium between nanodisc-bound and "free" or "micellar" copolymer chains in the solution. We believe that these findings can be exploited to optimize studies that involve the necessity of special copolymers, which would not only simplify the applications but also broaden the scope of polymer-based nanodiscs.

Toward Bridging a Gap Between Model and Natural Membranes for Biophysical Studies

To study their lipid interactions or obtain valuable structural information, membrane peptides or proteins need to be maintained in a lipid system as close as possible to their natural environment. Model membranes are employed in a variety of biophysical techniques such as nuclear magnetic resonance (NMR). However, developing a method that could allow peptide extraction and reconstitution in a native or near-native membrane would be an asset. In this work, we propose an approach inspired by bicelles, which can orient in the magnetic field. We prepared magnetically-orientable phosphatidylcholine (PC) liposomes using the membrane softening detergent Tween80 (polyoxyethylene sorbitan monooleate) which is also used to extract soluble and membrane-bound proteins. Our 31P and 2H solid-state NMR results show that TWeen80 mixed to DMPC and DPPC bilayers leads to elongated oriented structures which align over a wide range of lipid-to-detergent molar ratios (2<q<13) and temperatures (25-80oC). This can be ascribed to the miscibility of Tween80 and PC molecules, as shown by the thermotropic behavior of these mixture in infrared spectroscopy. We show that the orientation with respect to the magnetic field direction can be flipped from parallel to perpendicular by addition of lanthanides ions. Using a 15N-labeled model KALP transmembrane peptide, 15N solid-state NMR spectra showed that Tween80-based PC membranes can reveal a peptide orientation, thus providing valuable data for structural determination. Altogether, our work shows that Tween80 could be exploited for membrane protein extraction, reconstitution and structural studies without requiring to a detergent removal step. This approach has the potentiel to be used with more complex membrane bilayers including native bological membranes.

Disulfide Chaperone Knockouts Facilitate Double Spin Labelling of an Outer Membrane Transporter For in Vitro EPR Studies

The outer membrane (OM) of Gram-negative bacteria contains a number of porins and transporters that are crucial for bacterial growth and survival. These transporters include TonB-dependent transporters (TBDTs) that facilitate the active uptake of trace metals and complex carbohydrates, and are critical for the microbiome as well as the survival and virulence of many pathogens. During active transport, TBDTs couple with the trans-periplasmic protein, TonB and utilize energy from the inner membrane (IM) proton motive force. However, the exact mechanism of transport is not well understood. Recently, we have shown that it is possible to examine the structure of BtuB, the E. coli vitamin B12 transporter, using Electron Paramagnetic Resonance (EPR) spectroscopy in intact cells. Distance measurements using pulse EPR require that pairs of labels be placed within BtuB, which was accomplished by site-directed spin labelling of cysteines. in cells, this required the use of a knockout of the Dsb system that functions to oxidize pairs or cysteines. Using a dsbA- strain, we have shown that the protein appears to have a different configuration in vivo when compared to earlier work of reconstituted BtuB in liposomes. While comparing the in vivo and in vitro systems, we discovered that cysteine double mutants’ in vitro labeling efficiencies are poor when expressed in a wild-type strain. in contrast, we were able to efficiently spin label purified BtuB expressed in dsbA- strain. These results indicate that knocking out the Dsb system also promotes efficient double labeling for reconstituted membrane proteins that are isolated from the OM and facilitate the studies of BtuB in both in vivo and in vitro conditions.

Hybrid lipid/block copolymer vesicles display broad phase coexistence region

The fluidity and polar environment of ~100 nm hybrid vesicles combining dipalmitoylphosphatidylcholine (DPPC) and poly(1,2-butadiene)-block-polyethylene oxide (PBd-PEO, average molecular weight 950 g/mol) were studied upon vesicle heating using the fluorescence spectroscopy techniques of DPH anisotropy and laurdan generalized polarization (GP). These techniques indicated PBd-PEO membranes are less ordered than solid DPPC, but slightly more ordered than fluid DPPC or dioleoylphosphatidylcholine (DOPC) membranes. We find the DPH anisotropy values are less than expected from additivity of the components' anisotropies in the fluid phase mixture of DPPC and PBd-PEO, inferring that DPPC strongly fluidizes the PBd-PEO. We use transitions in DPH anisotropy and laurdan GP to create a temperature/composition phase diagram for DPPC/PBd-PEO which we find displays a significantly broader solid/fluid phase coexistence region than DPPC/DOPC, showing that DPPC partitions less readily into fluid PBd-PEO than into fluid DOPC. The existence of a broad solid/fluid phase coexistence region in DPPC/PBd-PEO vesicles is verified by Förster resonance energy transfer results and the visualization of phase separation in giant unilamellar vesicles containing up to 95% PBd-PEO and a single phase in 100% PBd-PEO vesicles at room temperature. These results add to the limited knowledge of phase behavior and phase diagrams of hybrid vesicles, and should be useful in understanding and tailoring membrane surface architecture toward biomedical applications such as drug delivery or membrane protein reconstitution.

Reconstitution of Detergent-Solubilized Membrane Proteins into Proteoliposomes and Nanodiscs for Functional and Structural Studies.

Reconstitution of detergent-solubilized membrane proteins into phospholipid bilayers allows for functional and structural studies under close-to-native conditions that greatly support protein stability and function. Here we outline the detailed steps for membrane protein reconstitution to result in proteoliposomes and nanodiscs. Reconstitution can be achieved via a number of different strategies. The protocols for preparation of proteoliposomes use detergent removal via dialysis or via nonpolar polystyrene beads, or a mixture of the two methods. In this chapter, the protocols for nanodiscs apply polystyrene beads only. Proteoliposome preparation methods allow for substantial control of the lipid-to-protein ratio, from minimal amounts of phospholipid to high concentrations, type of phospholipid, and mixtures of phospholipids. In addition, dialysis affords a fairly large degree of control and variation of parameters such as rate of reconstitution, temperature, buffer conditions, and proteoliposome size. For the nanodisc approach, which is highly advantageous for ensuring equal access to both membrane sides of the protein as well as fast reconstitution of only a single membrane protein into a well-defined bilayer environment in each nanodisc, the protocols outline how a number of these parameters are more restricted in comparison to the proteoliposome protocols.

A native cell membrane nanoparticles system allows for high-quality functional proteoliposome reconstitution

Proteoliposomes mimic the cell membrane environment allowing for structural and functional membrane protein analyses as well as antigen presenting and drug delivery devices. To make proteoliposomes, purified functional membrane proteins are required. Detergents have traditionally been used for the first step in this process. However, they can irreversibly denature or render membrane proteins unstable, and the necessary removal of detergents after reconstitution can decrease proteoliposome yields. The recently developed native cell membrane nanoparticles (NCMN) system has provided a variety of detergent-free alternatives for membrane protein preparation for structural biology research. Here we attempt to employ the MCMN system for the functional reconstitution of channels into proteoliposomes. NCMN polymers NCMNP1-1 and NCMNP7-1, members of a NCMN polymer library that have been successful in extraction and affinity purification of a number of intrinsic membrane proteins, were selected for the purification and subsequent reconstitution of three bacterial channels: KcsA and the mechanosensitive channels of large and small conductance (MscL and MscS). We found that channels in NCMN particles, which appeared to be remarkably stable when stored at 4 °C, can be reconstituted into bilayers by simply incubating with lipids. We show that the resulting proteoliposomes can be patched for electrophysiological studies or used for the generation of liposome-based nanodevices. In sum, the findings demonstrate that the NCMN system is a simple and robust membrane protein extraction and reconstitution approach for making high-quality functional proteoliposomes that could significantly impact membrane protein research and the development of nanodevices.

The application of Poisson distribution statistics in ion channel reconstitution to determine oligomeric architecture.

During reconstitution, membrane proteins are randomly inserted into liposomes according to Poisson distribution statistics. When the protein to lipid ratios in the reconstitution mixture are varied systematically, the characteristics of this statistical capture permit inferences about the proteins themselves, such as the number of subunits that assemble into a single functional unit. This chapter describes the Poisson distribution as applied to the reconstitution of membrane proteins into proteoliposomes and focuses on an application whereby this statistical behavior is used to determine the number of ion channel subunits that assemble into a functional pore. Practical considerations for performing these experiments are emphasized. Harnessing Poisson dilution statistics provides a function-based method to determine ion channel oligomerization, complementing other biophysical, biochemical, or structural approaches.

Membrane Protein Modified Electrodes in Bioelectrocatalysis

Transmembrane proteins involved in metabolic redox reactions and photosynthesis catalyse a plethora of key energy-conversion processes and are thus of great interest for bioelectrocatalysis-based applications. The development of membrane protein modified electrodes has made it possible to efficiently exchange electrons between proteins and electrodes, allowing mechanistic studies and potentially applications in biofuels generation and energy conversion. Here, we summarise the most common electrode modification and their characterisation techniques for membrane proteins involved in biofuels conversion and semi-artificial photosynthesis. We discuss the challenges of applications of membrane protein modified electrodes for bioelectrocatalysis and comment on emerging methods and future directions, including recent advances in membrane protein reconstitution strategies and the development of microbial electrosynthesis and whole-cell semi-artificial photosynthesis.

Membrane properties of ether-type phosphatidylcholine bearing partially fluorinated C18-monoacetylenic chains and their applicability to membrane protein reconstitution matrices

To examine the applicability of fluorinated membrane-forming phospholipids to reconstitution matrices for functional membrane proteins, the membrane properties of a synthetic ether-type phosphatidylcholine (PC) bearing partially fluorinated C18-monoacetylenic (9-octadecynyl) chains, DF8CCH8PC, were compared with those of its non-fluorinated counterpart, DH8CCH8PC. Light-harvesting complex 2 (LH2) and the light-harvesting 1‒reaction center core complex (LH1-RC) isolated from purple photosynthetic bacteria were employed as probe membrane proteins to evaluate the extent to which their reconstitution into DF8CCH8PC membranes could proceed. DF8CCH8PC formed more expanded and more stable fluid monolayers than DH8CCH8PC at the air-water interface at 25 °C; the former PC molecule occupied an area of ca. 0.70 nm2 at a collapse pressure, πc, of 52 mN/m, while the latter occupied an area of ca. 0.55 nm2 at a πc of 45 mN/m. In contrast, the molecular motion detected using fluorescent probes was much more restricted in DF8CCH8PC bilayers than in DH8CCH8PC ones. Although the reconstitution efficiencies of both LH2 and LH1-RC into DF8CCH8PC bilayers were lower than those into DH8CCH8PC bilayers, the membrane proteins incorporated into DF8CCH8PC bilayers showed increased thermostability. The increased thermostability of these proteins in fluorinated PC membranes might be due to the restricted molecular motion in the hydrophobic chains. The results of this study suggest that partially fluorinated PCs can be useful materials for the construction of lipid‒functional membrane protein assemblies including large membrane protein complexes, such as LH1-RC, for biotechnological applications.

Combinatorial Strategy for Studying Biochemical Pathways in Double Emulsion Templated Cell-Sized Compartments.

Cells rely upon producing enzymes at precise rates and stoichiometry for maximizing functionalities. The reasons for this optimal control are unknown, primarily because of the interconnectivity of the enzymatic cascade effects within multi-step pathways. Here, an elegant strategy for studying such behavior, by controlling segregation/combination of enzymes/metabolites in synthetic cell-sized compartments, while preserving vital cellular elements is presented. Therefore, compartments shaped into polymer GUVs are developed, producing via high-precision double-emulsion microfluidics that enable: i) tight control over the absolute and relative enzymatic contents inside the GUVs, reaching nearly 100% encapsulation and co-encapsulation efficiencies, and ii) functional reconstitution of biopores and membrane proteins in the GUVs polymeric membrane, thus supporting in situ reactions. GUVs equipped with biopores/membrane proteins and loaded with one or more enzymes are arranged in a variety of combinations that allow the study of a three-step cascade in multiple topologies. Due to the spatiotemporal control provided, optimum conditions for decreasing the accumulation of inhibitors are unveiled, and benefited from reactive intermediates to maximize the overall cascade efficiency in compartments. The non-system-specific feature of the novel strategy makes this system an ideal candidate for the development of new synthetic routes as well as for screening natural and more complex pathways.

Microfluidic Formation of Honeycomb-Patterned Droplets Bounded by Interface Bilayers via Bimodal Molecular Adsorption.

Assembled water-in-oil droplets bounded by lipid bilayers are used in synthetic biology as minimal models of cell tissue. Microfluidic devices successfully generate monodispersed droplets and assemble them via droplet interface bilayesr (DIB) formation. However, a honeycomb pattern of DIB-bounded droplets, similar to epithelial tissues, remains unrealized because the rapid DIB formation between the droplets hinders their ability to form the honeycomb pattern. In this paper, we demonstrate the microfluidic formation of a honeycomb pattern of DIB-bounded droplets using two surfactants with different adsorption rates on the droplet surface. A non-DIB forming surfactant (sorbitan monooleate, Span 80) was mixed with a lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, PC), whose adsorption rate on the droplet surface and saturated interfacial tension were lower than those of Span 80. By changing the surfactant composition, we established the conditions under which the droplets initially form a honeycomb pattern and subsequently adhere to each other via DIB formation to minimize the interfacial energy. In addition, the reconstituted membrane protein nanopores at the DIBs were able to transport molecules. This new method, using the difference in the adsorption rates of two surfactants, allows the formation of a honeycomb pattern of DIB-bounded droplets in a single step, and thus facilitates research using DIB-bounded droplet assemblies.

Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

Membrane proteins can be reconstituted in polymer-encased nanodiscs for studies under near-physiological conditions and in the absence of detergents, but traditional styrene-maleic acid copolymers used for this purpose suffer severely from buffer incompatibilities. We have recently introduced zwitterionic styrene-maleic amide copolymers (zSMAs) to overcome this limitation. Here, we compared the extraction and reconstitution of membrane proteins into lipid nanodiscs by a series of zSMAs with different styrene:maleic amide molar ratios, chain sizes, and molecular weight distributions. These copolymers solubilize, stabilize, and support membrane proteins in nanodiscs with different efficiencies depending on both the structure of the copolymers and the membrane proteins.

Use of a Poly‐γ‐Glutamic Acid‐Derived Amphipathic Polypeptide for the Reconstitution of Membrane Proteins

APG assisted stabilization and reconstitution of membrane proteins.

Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations.

Pichia pastoris is a eukaryotic microorganism reputed for its ability to mass-produce recombinant proteins, including integral membrane proteins, for various applications. This article details a series of protocols that progress towards the production of integral membrane proteins, their extraction and purification in the presence of detergents, and their eventual reconstitution in lipid nanoparticles. These basic procedures can be further optimized to provide integral membrane protein samples that are compatible with a number of structural and/or functional investigations at the molecular level. Each protocol provides general guidelines, technical hints, and specific recommendations, and is illustrated with case studies corresponding to several representative mammalian proteins. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Production of membrane proteins in a P. pastoris recombinant clone using methanol induction Basic Protocol 2: Preparation of whole-membrane fractions Alternate Protocol 1: Preparation of yeast protoplasts Basic Protocol 3: Extraction of membrane proteins from whole-membrane fractions Basic Protocol 4: Purification of membrane proteins Alternate Protocol 2: Purification of membrane proteins from yeast protoplasts Alternate Protocol 3: Simultaneous protoplast preparation and membrane solubilization for purification of membrane proteins Basic Protocol 5: Reconstitution of detergent-purified membrane proteins in lipid nanoparticles.

Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes.

Enzyme-mediated proton transport across biological membranes is critical for many vital cellular processes. pH-sensitive fluorescent dyes are an indispensable tool for investigating the molecular mechanism of proton-translocating enzymes. Here, we present a novel strategy to entrap pH-sensitive probes in the lumen of liposomes that has several advantages over the use of soluble or lipid-coupled probes. In our approach, the pH sensor is linked to a DNA oligomer with a sequence complementary to a second oligomer modified with a lipophilic moiety that anchors the DNA conjugate to the inner and outer leaflets of the lipid bilayer. The use of DNA as a scaffold allows subsequent selective enzymatic removal of the probe in the outer bilayer leaflet. The method shows a high yield of insertion and is compatible with reconstitution of membrane proteins by different methods. The usefulness of the conjugate for time-resolved proton pumping measurements was demonstrated by using two large membrane protein complexes.

New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins.

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

Mixed hybrid bilayer lipid membranes on mechanically polished titanium surface

Mixed self-assembled monolayers of octadecyltrichlorosilane (OTS) and methyltrichlorosilane (MTS) were deposited via simple silanization procedure on a mechanically polished titanium surface. The monolayers act as molecular anchors for mixed hybrid bilayer lipid membranes (mhBLM) which were accomplished via vesicle fusion. A variation of the MTS concentration in silanization solutions significantly affects properties of mhBLMs composed of a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Chol). The bilayers become less insulating following an increase of the MTS content. On the other hand, an increase of the MTS concentration provides flexibility of the mhBLM membranes necessary for the functional reconstitution of membrane proteins. The optimal molar ratio of MTS in silanization solution is 40% providing anchors for intact mhBLMs as confirmed by their specific capacitance of 0.86 μF cm−2. We found that the bilayers containing 40% (mol) of cholesterol bind cholesterol dependent pneumolysin (PLY). However, we did not observe functional reconstitution of PLY. While α-hemolysin almost fully disrupts mhBLMs assembled from 100% diphytanoyl. An important advantage of the titanium/OTS/MTS molecular anchor systems is their ability of repetitive regeneration of phospholipid bilayers without losing functional properties as demonstrated in the current study. This creates a possibility for the multiple-use phospholipid membrane biosensors which have a potential of decreasing the cost of such electrochemical/electroanalytical devices.