Erik Henrich

Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase

Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.

Membrane protein production in Escherichia coli cell‐free lysates

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.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

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.

Hydrophobic supplements in cell‐free systems: Designing artificial environments for membrane proteins

Membrane proteins (MPs) are of central interest for the pharmaceutical industry but their production is usually a challenging task. The complex folding mechanisms of newly synthesized MPs often require interactions with specific compounds for improved stability. Conditions for the production of high‐quality samples are therefore difficult to predict and frequently cannot be provided in conventional protein expression platforms. Cell‐free (CF) biosynthetic systems allow, in contrast to living cells, nonrestricted access to the protein production machinery. Reaction conditions can be adjusted according to particular requirements and modified by supplementing single additives or even cocktails of compounds. These options have initiated completely new research fields for the cotranslational stabilization and folding of MPs in artificial environments. Based on established and efficient CF production protocols, a recent focus was to explore and define suitable supplements for CF expression reactions that are useful for the generation of high‐quality MP samples. Besides classical detergents and lipids, a variety of new compounds with interesting properties have been discovered and synthesized. We compile the currently available toolbox for MP solubilization in CF systems and summarize new developments and perspectives for the directed modulation of CF biosynthetic environments.

Crystal Structure of a PCP/Sfp Complex Reveals the Structural Basis for Carrier Protein Posttranslational Modification

Phosphopantetheine transferases represent a class of enzymes found throughout all forms of life. From a structural point of view, they are subdivided into three groups, with transferases from group II being the most widespread. They are required for the posttranslational modification of carrier proteins involved in diverse metabolic pathways. We determined the crystal structure of the group II phosphopantetheine transferase Sfp from Bacillus in complex with a substrate carrier protein in the presence of coenzyme A and magnesium, and observed two protein-protein interaction sites. Mutational analysis showed that only the hydrophobic contacts between the carrier proteins second helix and the C-terminal domain of Sfp are essential for their productive interaction. Comparison with a similar structure of a complex of human proteins suggests that the mode of interaction is highly conserved in all domains of life.

Screening for lipid requirements of membrane proteins by combining cell-free expression with nanodiscs.

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.

Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology

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. DOI: http://dx.doi.org/10.7554/eLife.20954.001

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins

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.

Co-translational Stabilization of Insoluble Proteins in Cell-Free Expression Systems

Precipitation, aggregation, and inclusion body (IB) formation are frequently observed problems upon overexpression of recombinant proteins. The open accessibility of cell-free reactions allows addressing such critical steps by the addition of protein stabilizers such as chemical chaperones or detergents directly into the expression reactions. This approach could therefore reduce or even prevent initial protein precipitation already in the translation environment. The strategy might be considered to generally improve protein sample quality and to rescue proteins that are difficult to refold from IBs or from aggregated precipitates. We describe a protocol for the co-translational stabilization of difficult proteins by their expression in the presence of supplements such as alcohols, poly-ions, or detergents. We compile potentially useful compounds together with their recommended stock and working concentrations. Examples of screening experiments in order to systematically identify compounds or compound mixtures that stabilize particular proteins of interest are given. The method can primarily be considered for the production of unstable soluble proteins or of membrane proteins containing larger soluble domains.

Membrane Protein Quality Control in Cell-Free Expression Systems: Tools, Strategies and Case Studies

Cell-free expression has become an established platform with still rapidly expanding perspectives for the efficient production of membrane proteins. Applications and diversity of successfully synthesized targets, as well as the variety of cell-free expression tools, extract sources and reaction modifications are continuously increasing. Its modular nature, the elimination of expression problems caused by complex cell physiologies and its simplified reaction design secures cell-free expression a unique and exceptional position among the currently available protein production pipelines. Numerous protocols and case studies for the production of high-quality samples of diverse types of membrane proteins such as transporters, G -protein-coupled receptors, channels, porins, or membrane-integrated enzymes as well as of even large multi-subunit assemblies are now available.