Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in ...conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as ...substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have ...been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.
Nanodiscs that hold a lipid bilayer surrounded by a boundary of scaffold proteins have emerged as a powerful tool for membrane protein solubilization and analysis. By combining nanodiscs and ...cell-free expression technologies, even completely detergent-free membrane protein characterization protocols can be designed. Nanodiscs are compatible with various techniques, and due to their bilayer environment and increased stability, they are often superior to detergent micelles or liposomes for membrane protein solubilization. However, transport assays in nanodiscs have not been conducted so far, due to limitations of the two-dimensional nature of nanodisc membranes that offers no compartmentalization. Here, we study Krokinobacter eikastus rhodopsin-2 (KR2), a microbial light-driven sodium or proton pump, with noncovalent mass-spectrometric, electrophysiological, and flash photolysis measurements after its cotranslational insertion into nanodiscs. We demonstrate the feasibility of adsorbing nanodiscs containing KR2 to an artificial bilayer. This allows us to record light-induced capacitive currents that reflect KR2’s ion transport activity. The solid-supported membrane assay with nanodisc samples provides reliable control over the ionic condition and information of the relative ion activity of this promiscuous pump. Our strategy is complemented with flash photolysis data, where the lifetimes of different photointermediates were determined at different ionic conditions. The advantage of using identical samples to three complementary approaches allows for a comprehensive comparability. The cell-free synthesis in combination with nanodiscs provides a defined hydrophobic lipid environment minimizing the detergent dependence often seen in assays with membrane proteins. KR2 is a promising tool for optogenetics, thus directed engineering to modify ion selectivity can be highly beneficial. Our approach, using the fast generation of functional ion pumps incorporated into nanodiscs and their subsequent analysis by several biophysical techniques, can serve as a versatile screening and engineering platform. This may open new avenues for the study of ion pumps and similar electrogenic targets.
Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution nuclear magnetic resonance (NMR) spectroscopy of integral membrane proteins (IMPs). Despite varied challenges ...to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression. Our results demonstrate appreciable gains in NMR spectral quality while preserving lipid-IMP contacts. We validate the approach on the detergent-sensitive LspA, which finally allowed us to perform high-quality triple-resonance NMR experiments for structural studies. Our strategy of producing bicelles from nanodiscs comprehensively avoids detergent during expression and preparation and is suitable for solution NMR spectroscopy of lipid-IMP complexes.
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•A strategy to produce bicelles from integral membrane proteins in nanodisc bilayers•Suitable for solution NMR studies of detergent-sensitive membrane proteins•Vast improvements in signal intensity allow high-resolution NMR spectroscopy
Laguerre et al. show that nanodisc bilayers can be peeled away from embedded membrane proteins by detergent titration to make bicelles. Avoiding initial detergent solubilization, this method preserves lipid contacts and functional folds of detergent-sensitive membrane proteins. The resulting improvements in spectral intensity facilitate high-resolution NMR spectroscopy for structure determination.
Cell-free (CF) protein expression has emerged as one of the most efficient production platforms for membrane proteins. Central bottlenecks prevalent in conventional cell-based expression systems such ...as mistargeting, inclusion body formation, degradation as well as product toxicity can be addressed by taking advantage of the reduced complexity of CF expression systems. However, the open accessibility of CF reactions offers the possibility to design customized artificial expression environments by supplying synthetic hydrophobic compounds such as micelles or membranes of defined composition. The open nature of CF systems therefore generally allows systematic screening approaches for the identification of efficient cotranslational solubilization environments of membrane proteins. Synergies exist in particular with the recently developed nanodisc (ND) technology enabling the synthesis of stable and highly soluble particles containing membrane discs of defined composition. Specific types of lipids frequently modulate folding, stability, and activity of integrated membrane proteins. One recently reported example are phospho-MurNAc-pentapeptide (MraY) translocases that catalyze a crucial step in bacterial peptidoglycan biosynthesis making them interesting as future drug targets. Production of functionally active MraY homologues from most human pathogens in conventional cellular production systems was so far not successful due to their obviously strict lipid dependency for functionally folding. We demonstrate that the combination of CF expression with ND technologies is an efficient strategy for the production of folded MraY translocases, and we present a general protocol for the rapid screening of lipid specificities of membrane proteins.
Native mass spectrometry is applied for the investigation of proteins and protein complexes worldwide. The challenge in native mass spectrometry is maintaining the features of the proteins of ...interest, such as oligomeric state, bound ligands, or the conformation of the protein complex, during transfer from solution to gas phase. This is an essential prerequisite to allow conclusions about the solution state protein complex, based on the gas phase measurements. Therefore, soft ionization techniques are required. Widely used for the analysis of protein complexes are nanoelectro spray ionization (nESI) mass spectrometers. A newer ionization method is laser induced liquid bead ion desorption (LILBID), which is based on the release of protein complexes from solution phase via infrared (IR) laser desorption. We use both methods in our lab, depending on the requirements of the biological system we are interested in. Here we benchmark the performance of our LILBID mass spectrometer in comparison to a nESI instrument, regarding sample conditions, buffer and additive tolerances, dissociation mechanism and applicability towards soluble and membrane protein complexes.
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•New experiments separate non-unique residue pairs in selectively labeled proteins.•Fewer samples are needed for combinatorial-labeling based backbone assignments.•Exclusively ...recording 2D spectra relaxes measurement time requirements.•Complete protein amide assignment without uniform labeling appears feasible.
Combinatorial selective isotope labeling is a valuable tool to facilitate polypeptide backbone resonance assignment in cases of low sensitivity or extensive chemical shift degeneracy. It involves recording of 15N-HSQC and 2D HN-projections of triple-resonance spectra on a limited set of samples containing different combinations of labeled and unlabeled amino acid types. Using labeling schemes in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes – individually as well as simultaneously – usually yields abundant amino-acid type information of consecutive residues i and i − 1. Although this results in a large number of anchor points that can be used in the sequential assignment process, for most amide signals the exact positioning of the corresponding residue the polypeptide sequence still relies on matching intra- and interresidual 13C chemical shifts obtained from 3D spectra. An obvious way to obtain more sequence-specific assignments directly with combinatorial labeling would be to increase the number of samples. This is, however, undesirable because of increased sample preparation efforts and costs. Irrespective of the number of samples, unambiguous assignments cannot be accomplished for i − 1/i pairs that are not unique in the sequence. Here we show that the ambiguity for non-unique pairs can be resolved by including information about the labeling state of residues i + 1 and i − 2. Application to a 35-residue peptide resulted in complete assignments of all detectable signals in the 15N HSQC which, due to its repetitive sequence and 13C chemical shift degeneracies, was difficult to achieve by other means. For a medium-sized protein (165 residues, rotational correlation time 8.2 ns) the improved protocol allowed the extent of backbone amide assignment to be expanded to 88% solely using a suite of 2D 1H-15N correlated spectra.
Cotranslational insertion of membrane proteins into defined nanoparticle membranes has been developed as an efficient process to produce highly soluble samples in native-like environments and to ...study lipid-dependent effects on protein structure and function. Numerous examples of the structural and functional characterization of transporters, ion channels, or G-protein-coupled receptors in cotranslationally formed nanodisc complexes demonstrate the versatility of this approach, although the basic underlying mechanisms of membrane insertion are mainly unknown. We have revealed the first aspects of the insertion of proteins into nanodiscs by combining cell-free expression, noncovalent mass spectrometry, and NMR spectroscopy. We provide evidence of cooperative insertion of homo-oligomeric complexes and demonstrate the possibility to modulate their stoichiometry by modifying reaction conditions. Additionally, we show that significant amounts of lipid are released from the nanodiscs upon insertion of larger protein complexes.
The integral synaptic vesicle protein SV31 has been shown to bind divalent cations. Here, we demonstrate that SV31 protein synthesized within a cell‐free system binds Zn2+ and to a lower extent Ni2+ ...and Cu2+ ions. Expression with Zn2+ stabilized the protein and increased solubility. SV31 was preferentially monomeric in detergent and revealed specific binding of Zn2+. When co‐translationally inserted into defined nanodisc bilayers, SV31 assembled into dimeric complexes, resulting in increased binding of Zn2+. Putative Zn2+‐binding motifs within SV31 comprise aspartic acid and histidine residues. Site‐directed mutagenesis of two conserved aspartic acid residues leads to a potent decrease in Zn2+ binding but did not affect dimerization. Chemical modification of histidine residues abolished some of the Zn2+‐binding capacity. We demonstrate proton‐dependent transport of Zn2+ as by accumulation of fluorescent FluoZin‐1 inside of SV31‐containing proteoliposomes. Transport activity has a Km value of 44.3 μM and required external Zn2+ and internal acidic pH. Our results demonstrate that the synaptic vesicle‐integral protein SV31 functions as a proton‐dependent Zn2+ transporter. SV31 may attribute specific and yet undiscovered functions to subsets of synapses.
The synaptic vesicle Zn2+‐binding protein SV31 functions as a proton‐dependent Zn2+ transporter. Dimeric state, binding activity as well as stability are favored if SV31 is expressed in a lipid environment such as nanodiscs. Site‐directed mutagenesis of homologues amino acid residues involved in Zn2+‐binding selected by comparison with a bacterial Zn2+ transporter revealed two aspartic residues crucial for transport.
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