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We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein 'remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced 1 by formation of the proposed a-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15 N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins.

Protein dynamics plays an important role in protein function. Many functionally important motions occur on the microsecond and low millisecond time scale and can be characterized by nuclear magnetic resonance relaxation experiments. We describe the different states of a peptidyl carrier protein (PCP) that play a crucial role in its function as a peptide shuttle in the nonribosomal peptide synthetases of the tyrocidine A system. Both apo-PCP (without the bound 4'-phosphopantetheine cofactor) and holo-PCP exist in two different stable conformations. We show that one of the apo conformations and one of the holo conformations are identical, whereas the two remaining conformations are only detectable by nuclear magnetic resonance spectroscopy in either the apo or holo form. We further demonstrate that this conformational diversity is an essential prerequisite for the directed movement of the 4'-PP cofactor and its interaction with externally acting proteins such as thioesterases and 4'-PP transferase.

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.

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.

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial a-helical multidrug transporter, EmrE, the b-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-snglycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a b-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent. (20)-oleyl-ether; CE, continuous exchange; CF, cell-free; CMC, critical micellar concentration; C mic , micellar concentration; DDM, N-dodecyl-b-D-maltoside; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine; diC 6 PC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine; diC 8 PC, 1,2-dioctanoyl-sn-glycero-3-phosphocholine; DM, n-decyl-b-maltoside; DPC, dodecylphosphocholine; FM, feeding mixture; Genapol C-100, polyoxyethylene-(10)-dodecyl-ether; GPCR, G-protein coupled receptor; LMPG, 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; LPPG, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; MP, integral membrane protein; NP40, nonylphenyl polyethylene glycol; RM, reaction mixture; Thesit, polyethylene glycol 400 dedecyl ether; TMS, transmembrane segment; Triton X-100, polyethylene glycol P-1,1,3,3-tetramethyl-butylphenyl ether; Tween 20, polyoxyethylene sorbitan monolaurate 20.

The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function expression of the linked esaI gene, thus EsaR has no role in controlling coinducer synthesis. IntroductionBacteria express selected gene systems in a populationdependent manner by sensing self-produced, membranediffusible signals in a strategy called quorum sensing (QS) . The key elements of QS regulation in many Gram-negative bacteria are homologue proteins of LuxI, a N-acyl-homoserinelactone (AHL) signal synthase, and LuxR, an AHL-dependent response regulator. These two proteins control the expression of bioluminescence in the marine bacterium, Vibrio fischeri (Fuqua et al., 1994;Williams et al., 2000;Fuqua et al., 2001;Miller and Bassler, 2001;Withers et al., 2001). Alternative QS mechanisms, mediated by unrelated control factors, exist in other Gram-negative bacteria, most notably Vibrio harveyi (Bassler et al., 1994), and in several Grampositive organisms (Dunny and Leonard, 1997;Kleerebezem and Quadri, 2001). In general, QS governs the control of diverse phenotypes, each benefiting a bacterium in a specialized habitat (Whiteley et al., 1999;Pierson, 2000;Whitehead et al., 2001).Pantoea stewartii ssp. stewartii (P. stewartii) is the causative agent of Stewart's wilt disease in sweetcorn and leaf blight in maize. Disease symptoms develop when the bacterium produces large amounts of a capsular polysaccharide (CPS), which blocks the corn xylem vessels and induces necrotic lesions (Coplin et al., 1992). CPS synthesis is a QS-controlled phenotype governed by the LuxI and LuxR homologue proteins, EsaI and EsaR (von Bodman and Farrand, 1995). Disruption of the signal synthase gene, esaI, leads to parallel loss of AHL, CPS production, and virulence. In contrast, mutations in the esaR gene give a hypermucoid phenotype irrespective of AHL (von Bodman et al., 1998). The simplest explanation for these observations is that EsaR functions as a repressor of CPS synthesis and that derepression requires inducing levels of AHL. The functions required for CPS synthesis are encoded by an extensive cps gene system (Dolph et al., 1988). This gene system is closely related to the wza gene cluster encoding the synthesis of the group I capsules, colanic acid in Escherichia coli (Reeves et al., 1996), and amylovoran in Erwinia

A solved puzzle: The structure of the seven‐transmembrane‐helix proton pump proteorhodopsin obtained by solution NMR spectroscopy is based on NOE data combined with distance restraints derived from paramagnetic relaxation enhancement (see picture). Restraints from residual dipolar couplings improved the structural accuracy.