Tellurite-squarate driven assembly of a new family of nanoscale clusters based on (Mo2O2S2)2+

We report the preparation and characterization of a new family of four polyoxothiometalate (POTM) clusters, with varying size and complexity, based upon the dimeric [Mo2O2S2(H2O)6]2+ with the general formula (NMe4)aKb[(Mo2O2S2)c(TeO4)d(C4O4)e(OH)f] where a,b,c,d,e,f = {1,7,14,2,4,10} = 1, {Mo28Te2}; {2,26,36,4,10,48} = 2, {Mo72Te12}; {0,11,15,3,3,21} = 3, {Mo30Te3}; {2,6,12,2,4,16} = 4, {Mo24Te2}. The incorporation of tellurite anions allowed the fine tuning of the templation and bridging of the available building blocks leading to new topologies of increased complexity. The structural diversity of this family of compound, ranges from the highly symmetrical cross-shaped {Mo24Te2} to the stacked ring structure of {Mo72Te12} which is the largest chalcogen-containing POTM cluster reported so far. Also a detailed experimental analysis revealed that the pH isolation window extends form acidic to basic values. ESI-MS analyses not only confirmed the stability of this family in solution but also revealed the stability of the observed virtual building blocks

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of E.coli

Background: Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria. Methods: The ß-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the E.coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined. Results: Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold. Conclusions: The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. General Significance: We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors

Characterisation of components and mechanisms involved in redox-regulation of protein import into chloroplasts

The vast majority of chloroplast proteins is encoded in the nucleus and thus has to be posttranslationally imported into the organelle, a process that is facilitated by two multimeric protein machineries, the Toc and Tic complexes (translocon at the outer/inner envelope of chloroplasts). Regulation of protein import, e.g. by redox signals, is a crucial step to adapt the protein content to the biochemical requirements of the organelle. In particular, one subunit of the Tic complex, Tic62, has been proposed as a redox sensor, whose possible function is to regulate protein import by sensing and reacting to the redox state of the organelle. To elucidate a potential redox regulation of protein import, structural features, redox-dependent properties and the evolutional origin of Tic62 were investigated. The results show that Tic62 consists of two very different modules: the N-terminal part was found to be mainly -helical and possesses dehydrogenase activity in vitro. It is furthermore an evolutionary ancient domain, as it is highly conserved in all photosynthetic organisms from flowering plants to cyanobacteria and even green sulfur bacteria. In contrast to this, the C-terminus is largely disordered and interacts specifically with ferredoxin-NADP+ oxidoreductase (FNR), a key enzyme in photosynthetic electron transfer reactions. Moreover, this domain was found to exist only in flowering plants, and thus the full-length Tic62 protein seems to be one of the evolutionary youngest Tic components. The results of this study make also clear that Tic62 is a target of redox regulation itself, as its localization and interaction properties depend on the metabolic redox state: oxidized conditions lead to fast membrane binding and interaction with the Tic complex, whereas reduced conditions cause solubilization of Tic62 into the stroma and increased interaction with FNR. This novel shuttling behaviour indicates a dynamic composition of the Tic complex. The NADP+/NADPH ratio was furthermore found to be able to influence the import efficiency of many precursor proteins. Interestingly, the import of not all preproteins depends on the stromal redox state. Hence it was proposed that not a single stable Tic translocon exists, but several Tic subcomplexes with different subunit compositions, which might mediate the import of different precursor groups in a redox-dependent or -independent fashion. Another redox signal that was analyzed in regard to an impact on protein import is the reversible reduction of disulfide bridges, which was found to affect the channel and receptor proteins of the Toc complex. The import of all proteins that use the Toc translocon for entering the chloroplast was shown to be influenced by disulfide bridge formation. Thus it can be concluded that a variety of redox signals, acting both on the Toc and Tic complexes, are able to influence chloroplast protein import

Oxidative protein folding in the mitochondrial intermembrane space

Disulfide bond formation is a crucial step for oxidative folding and necessary for the acquisition of a protein's native conformation. Introduction of disulfide bonds is catalyzed in specialized subcellular compartments and requires the coordinated action of specific enzymes. The intermembrane space of mitochondria has recently been found to harbor a dedicated machinery that promotes the oxidative folding of substrate proteins by shuttling disulfide bonds. The newly identified oxidative pathway consists of the redox-regulated receptor Mia40 and the sulfhydryl oxidase Erv1. Proteins destined to the intermembrane space are trapped by a disulfide relay mechanism that involves an electron cascade from the incoming substrate to Mia40, then on to Erv1, and finally to molecular oxygen via cytochrome c. This thiol–disulfide exchange mechanism is essential for the import and for maintaining the structural stability of the incoming precursors. In this review we describe the mechanistic parameters that define the interaction and oxidation of the substrate proteins in light of the recent publications in the mitochondrial oxidative folding field

Model for eukaryotic tail-anchored protein binding based on the structure of Get3

The Get3 ATPase directs the delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER). TA-proteins are characterized by having a single transmembrane helix (TM) at their extreme C terminus and include many essential proteins, such as SNAREs, apoptosis factors, and protein translocation components. These proteins cannot follow the SRP-dependent co-translational pathway that typifies most integral membrane proteins; instead, post-translationally, these proteins are recognized and bound by Get3 then delivered to the ER in the ATP dependent Get pathway. To elucidate a molecular mechanism for TA protein binding by Get3 we have determined three crystal structures in apo and ADP forms from Saccharomyces cerevisae (ScGet3-apo) and Aspergillus fumigatus (AfGet3-apo and AfGet3-ADP). Using structural information, we generated mutants to confirm important interfaces and essential residues. These results point to a model of how Get3 couples ATP hydrolysis to the binding and release of TA-proteins

Structures of Sortase B from Staphylococcus aureus and Bacillus anthracis Reveal Catalytic Amino Acid Triad in the Active Site

Surface proteins attached by sortases to the cell wall envelope of bacterial pathogens play important roles during infection. Sorting and attachment of these proteins is directed by C-terminal signals. Sortase B of S. aureus recognizes a motif NPQTN, cleaves the polypeptide after the Thr residue, and attaches the protein to pentaglycine cross-bridges. Sortase B of B. anthracis is thought to recognize the NPKTG motif, and attaches surface proteins to m-diaminopimelic acid cross-bridges. We have determined crystal structure of sortase B from B. anthracis and S. aureus at 1.6 and 2.0 Å resolutions, respectively. These structures show a β-barrel fold with α-helical elements on its outside, a structure thus far exclusive to the sortase family. A putative active site located on the edge of the β-barrel is comprised of a Cys-His-Asp catalytic triad and presumably faces the bacterial cell surface. A putative binding site for the sorting signal is located nearby