Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

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.

BDNF/TrkB signaling plays critical roles in axonal outgrowth of neurons, the process of which requires the remodeling of the cytoskeleton structure, including microtubules and filamentous actin. However, the mechanism by which BDNF/TrkB signaling regulates cytoskeleton reorganization is still unclear. Here, we identified a novel interaction between LIMK1 and TrkB, which is required for the BDNF-induced axonal elongation. We demonstrated that BDNF-induced TrkB dimerization led to LIMK1 dimerization and transphosphorylation independent of TrkB kinase activity, which could further enhance the activation and stabilization of LIMK1. Moreover, activated LIMK1 translocated to the membrane fraction and phosphorylated its substrate cofilin, thus promoting actin polymerization and axonal elongation. Our findings provided evidence of a novel mechanism for the BDNF-mediated signal transduction leading to axonal elongation.

Extraction and purification of membrane proteins has for a long time represented a significant challenge. Polymer‐based extraction methods, like those using styrene maleic acid co‐polymers have provided a fertile approach to generate samples that include the local lipid environment surrounding the protein. However, the wide variety of different polymers now available provides a challenge to identify the optimal solution. In this study we develop and demonstrate a novel high‐throughput screening approach for rapid optimization of polymer solubilization agents and chromatography resins for membrane protein purification. Using this approach, we explore whether there are standard conditions that perform well for a range of membrane protein morphologies, sources and functions. These data show that no such standard conditions exist for either polymer solubilization agent or chromatography resin and that some combinations are rarely suitable for membrane protein purifications under these conditions, such as the use of TALON resin at a pH of 7.5 or SMALP300 in the Synthetic Nanodisc Screening Kit MINI kit. Instead, the use of the screening approach developed in this work is the best route to an optimal membrane protein preparation protocol.

Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy. A brief summary of alternative membrane protein solubilizing and stabilizing systems is also included. Protocols in this unit include the isolation and solubilization of biological membranes and phase separation; support protocols for detergent removal, detergent exchange, and the determination of critical micelle concentration using different methods are also included.

Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio-function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta- barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

Styrene maleic acid (SMA) polymers have proven to be very successful for the extraction of membrane proteins, forming SMA lipid particles (SMALPs), which maintain a lipid bilayer around the membrane protein. SMALP-encapsulated membrane proteins can be used for functional and structural studies. The SMALP approach allows retention of important protein-annular lipid interactions, exerts lateral pressure, and offers greater stability than traditional detergent solubilisation. However, SMA polymer does have some limitations, including a sensitivity to divalent cations and low pH, an absorbance spectrum that overlaps with many proteins, and possible restrictions on protein conformational change. Various modified polymers have been developed to try to overcome these challenges, but no clear solution has been found. A series of partially-esterified variants of SMA (SMA 2625, SMA 1440 and SMA 17352) has previously been shown to be highly effective for solubilisation of plant and cyanobacterial thylakoid membranes. It was hypothesised that the partial esterification of maleic acid groups would increase tolerance to divalent cations. Therefore, these partially-esterified polymers were tested for the solubilisation of lipids and membrane proteins, and their tolerance to magnesium ions. It was found that all partially esterified polymers were capable of solubilising and purifying a range of membrane proteins, but the yield of protein was lower with SMA 1440, and the degree of purity was lower for both SMA 1440 and SMA 17352. SMA 2625 performed comparably to SMA 2000. SMA 1440 also showed an increased sensitivity to divalent cations. Thus, it appears the interactions between SMA and divalent cations are more complex than proposed and require further investigation.

Detergents are amphiphilic compounds with crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state nuclear magnetic resonance (NMR) spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution/high-sensitivity spectra. The best quality NMR spectra use detergents with partial or complete deuteration, which eliminate interfering proton signals from the detergent itself and eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Another technique for which deuterated detergents have been crucial to obtain structural information about membrane proteins in solution is small-angle neutron scattering (SANS). Use of a deuterated detergent exploits the intrinsic neutron scattering length difference between hydrogen and deuterium, such that detergent is matched-out in D2O and only signal from the membrane protein remains visible in the scattering profile, simplifying data analysis. We provide an updated review on the properties, chemical synthesis and applications of detergents that are commercially available and/or that have been synthesized with partial or complete deuteration, and that have been used in NMR or SANS studies on membrane proteins. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). We also consider effects of deuteration, detergent screening and guidelines for detergent selection.

In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is, however, often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and co-purified during the purification process. In order to minimize this unwanted phenomenon, we devised a novel procedure in which MPs produced in Pichia pastoris are directly solubilized from whole cells instead of crude membrane preparation. This approach aims at favoring the extraction of correctly folded membrane proteins that have been targeted to the plasma membrane, limiting the solubilization of the misfolded proteins and protein aggregates that are stored in internal membrane compartments. The method described herewith is based on the formation of protoplasts through enzymatic treatment prior to protein solubilization. This chapter details a set of protocols going from yeast cell preparation and protein solubilization to purification using affinity and size exclusion chromatography.

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

Conformational transitions of maleic acid segment drive pH induced changes in SMA polymer structure and solubility

Molecular Simulations of Dodecyl-D-Maltoside Micelles in Water: Influence of the Headgroup Conformation and the Force Field Parameters

Single-domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell-surface display. However, for membrane proteins, the targets are generally detergent-solubilized, and there remains a need for Nb development methods against membrane proteins in a native-like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as styrene-maleic acid to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to four membrane-resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane-associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways. The selected Nbs showed high affinity and selectivity towards their target proteins with KD apparent values ranging from 15 nM to 200 nM, depending on the Nb-protein conjugate. In accordance with their tight binding, various Nb-protein complexes were found to be stable to size-exclusion chromatography purification. The Nbs were also amenable to sortase-mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high-affinity Nb against membrane proteins in SMALP will facilitate their widespread application in cell biology and biomedical applications.

BackgroundMembrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.Methodology/Principal FindingsAnionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.Conclusion/SignificanceThese compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.

Systematic Analyses of structure/function variability of viral K+ channels for the development of synthetic channels

Detergents are indispensable reagents for the extraction and solubilization of integral membrane proteins, but their removal from a reconstituted phospholipid-protein complex is usually desirable. In this paper, we describe a novel method in which the synthetic sugar esters 6-O-octanoyl-beta-D-glucose (OG) or 6-O-octanoyl-beta-D-mannose (OM) are used as detergents for both the isolation and the rapid reconstitution of the photosynthetic reaction center protein of Rhodobacter sphaeroides. Following solubilization of the reaction center with OG or OM and reconstitution of this protein in liposomes, a convenient removal of these detergents was achieved within less than two hours by hydrolytic cleavage of the sugar esters using immobilized lipases. Best results were achieved with lipase from Bacillus sp. immobilized on silica gel.