Christopher Hein

Structural Basis for Tail-Anchored Membrane Protein Biogenesis by the Get3-Receptor Complex

Docking of cytoplasmic and membrane receptors facilitates conformational changes that drive protein insertion. Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.

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.

Characterization of molecular interactions between ACP and halogenase domains in the curacin A polyketide synthase

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP(I)-ACP(II)-ACP(III)) within the CurA PKS module. We have determined the structure of the isolated holo-ACP(I) and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP(I), which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP(I) and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP(I)-ACP(II)-ACP(III) tridomain, our data provide insight into the specificity of the chlorination reaction.

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.

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

Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.

Insights into Cotranslational Membrane Protein Insertion by Combined LILBID-Mass Spectrometry and NMR Spectroscopy

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.

How to switch a master switch

The crystal structure of a nucleotide exchange factor in white blood cells reveals an autoinhibitory mechanism that reinforces the switch-like behaviour of the signalling protein Ras.