Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH 2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH 2 to PGE 2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.electron crystallography ͉ inflammation ͉ MAPEG ͉ membrane protein M icrosomal prostaglandin E synthase 1 (MPGES1) is the key enzyme in pathology related production of PGE 2 from cyclooxygenase (Cox) derived PGH 2 (1). The protein is a member of the MAPEG protein family, which includes 5-lipoxygenase activating protein (FLAP), leukotriene C 4 synthase (LTC4S), microsomal glutathione transferase (MGST)1, MGST2, and MGST3 (2, 3). MPGES1 is the most efficient PGES known and catalyzes the oxidoreduction of prostaglandin endoperoxide H 2 into PGE 2 with an apparent k cat /K m of 310 mM Ϫ1 s Ϫ1 [supporting information (SI) Fig. S1]. The enzyme equally well catalyses the oxidoreduction of endocannabinoids into prostaglandin glycerol esters (4) and PGG 2 into 15-hydroperoxy-PGE 2 (5). In addition, the enzyme confers low glutathione transferase and glutathione-dependent peroxidase activities (5). The biological significance of the latter activities remains unclear but is thought to reflect the close evolutionary distance to MGST1.MPGES1 protein expression levels are in most cases low, and proinflammatory stimuli induce its cellular expression and activity, which is prevented by corticosteroids (1, 6-8). The predominant source of PGH 2 seems derived from Cox-2, although Cox-1 may also contribute (9). Studies, mainly from disruption of the MPGES1 gene in mice, indicate key roles for MPGES1-generated PGE 2 in pathological conditions such as chronic inflammation, pain, fever, anorexia, atherosclerosis, stroke and tumorigenesis (10). Recently, a role for MPGES1 in regulating neonatal respiration was described in ref. 11. MPGES1 has been shown to be overexpressed in rheu...

Potassium channels ubiquitously exist in nearly all kingdoms of life and perform diverse but important functions. Since the first atomic structure of a prokaryotic potassium channel (KcsA, a channel from Streptomyces lividans) was determined, tremendous progress has been made in understanding the mechanism of potassium channels and channels conducting other ions. In this review, we discuss the structure of various kinds of potassium channels, including the potassium channel with the pore-forming domain only (KcsA), voltage-gated, inwardly rectifying, tandem pore domain, and ligand-gated ones. The general properties shared by all potassium channels are introduced first, followed by specific features in each class. Our purpose is to help readers to grasp the basic concepts, to be familiar with the property of the different domains, and to understand the structure and function of the potassium channels better.

Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.

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The molecular structure of Na,K-ATPase was determined by electron crystallography from two-dimensional crystals induced in purified membranes isolated from the outer medulla of pig kidney. The P2 type unit cell contains two protomers in the E(2) conformation, each of them with a size of 65 x 75 x 150 A(3). The alpha, beta, and gamma subunits in the membrane crystals were demonstrated in the crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as to the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains. One of these gives rise to an elongated projection onto the membrane plane, while the putative nucleotide binding and phosphorylation domains form compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit. Ten helices from the catalytic a subunit correspond to two groups of distinct densities in the transmembrane region. The structure of the lipid bilayer spanning part also suggests positions for the transmembrane helices from the beta and gamma subunits. The overall structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy is similar to the X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmic domains.

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25°in relation to each other, suggesting a role for global dynamics in dodecamer function.

The ion-coupled sugar membrane symporter or co-transporter melibiose permease (MelB), responsible for alpha-galactoside accumulation in Escherichia coli, is a representative member of the glycoside-pentoside- hexuronide family of the vast class of electrochemical potential-driven porters. Pure solubilized preparations of a MelB recombinant protein were subjected to two-dimensional crystallization trials and several crystal forms were observed. Two of these appeared as large wide tubes suitable for analysis by electron crystallography. Flattened tubes on carbon support film, embedded in amorphous ice prior to electron cryomicroscopy, showed two-sided plane group symmetries P12(1) or P222(1), with unit cell dimensions a = 89.9 A, b = 51.6 A, gamma = 91.9 degrees and a = 188.9 A, b = 48.8 A, gamma = 90 degrees, respectively. The projection map from the P222(1 )crystals at 8 A resolution displayed an asymmetric protein unit consisting of two domains lining a central and curve-shaped cleft. Together, the MelB monomer could host the 12 predicted transmembrane alpha-helices. Overall, the MelB helix packing arrangement compared more favorably with that of the Na(+)/H(+) antiporter NhaA than that of the oxalate antiporter.