Production of Membrane Proteins in Pseudomonas stutzeri.

Functional and structural studies on membrane proteins are often hampered by insufficient yields, misfolding and aggregation during the production and purification process. Escherichia coli is the most commonly used expression host for the production of recombinant prokaryotic integral membrane proteins. However, in many cases expression hosts other than E. coli are more appropriate for certain target proteins. Here, we report a convenient, systematically developed expression system using the γ-proteobacterium Pseudomonas stutzeri as an alternative production host for over-expression of integral membrane proteins. P. stutzeri can be easily and inexpensively cultured in large quantities. The Pseudomonas expression vectors are designed for inducible expression of affinity-tagged fusion proteins controlled by the PBAD promoter. This chapter provides detailed protocols of the different steps required to successfully produce and isolate recombinant membrane proteins with high yields in P. stutzeri.

Cryo-EM structures of pentameric autoinducer-2 exporter from Escherichia coli reveal its transport mechanism.

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for both intra- and inter-species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI-2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI-2 exporter superfamily, has been shown to export AI-2. Here, we report the cryogenic electron microscopic structures of two AI-2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI-2 exporter exists as a homo-pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI-2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator-type transport mechanism.

Rational engineering of Luminiphilus syltensis (R)-selective amine transaminase for the acceptance of bulky substrates.

Despite the plethora of information on (S)-selective amine transaminases, the (R)-selective ones are still not well-studied; only a few structures are known to date, and their substrate scope is limited, apart from a few stellar works in the field. Herein, the structure of Luminiphilus syltensis (R)-selective amine transaminase is elucidated to facilitate engineering towards variants active on bulkier substrates. The V37A variant exhibited increased activity towards 1-phenylpropylamine and to activity against 1-butylamine. In contrast, the S248 and T249 positions, located on the ß-turn in the P-pocket, seem crucial for maintaining the activity of the enzyme.

Expression, purification and characterization of the IcmG and IcmK proteins of the type IVB secretion system from Coxiella burnetii.

Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterial pathogen. Studies on Coxiella have shown that a type IVB secretion system (T4BSS) contributes to the establishment of the infection by transferring protein molecules. In this report, we focus on two core proteins of the Coxiella T4BSS, namely the IcmG/DotF protein (CBU_1626) and the IcmK/DotH protein (CBU_1628). Here we present a method for the recombinant expression of IcmG and IcmK in E. coli. IcmG was purified by Strep-Tactin affinity chromatography and size exclusion chromatography, while for the purification of IcmK an additional anion exchange chromatography step was introduced. The yields of the purified IcmG and IcmK proteins were 1.2 mg/L and 3 mg/L, respectively. The purified proteins showed predominant band on SDS-PAGE gel of 37 kDa for the IcmG and 40 kDa for the IcmK. Protein folding is confirmed by circular dichroism spectroscopy. The dynamic light scattering experiment indicated that IcmG and IcmK existed in a homogenous form. Further Blue native PAGE indicates the presences of a monomeric form for the IcmK and IcmG. Our work lays the basis for functional exploration and structural determination of IcmG and IcmK proteins of Coxiella's secretion system.

Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus.

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Šand revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.

Inward-facing conformation of a multidrug resistance MATE family transporter.

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters.

Comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics.

Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.