Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in ...the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms.
A one-step procedure using carbodiimide condensation was proposed for controlled functionalization of watersoluble semiconductor quantum dots (QDs) with azide groups. The changes of functional group ...surface density and ζ-potential could be controlled during modifi cation of anionic and analogous zwitterionic polymer-encapsulated CdSe/ZnS core–shell QDs. Ethanolamine-O-sulfate, aminotetraethylene glycol (H
2
N–TEG–OH), and aminotetraethylene glycol azide (H
2
N–TEG–N
3
) were used as modifiers. Functionalization was investigated by studying the conjugation kinetics of organic dye JOE to QDs using strain-promoted azide-alkyne cycloaddition (SPAAC). The reactivity of QDs in SPAAC was nearly proportional to the relative density of surface azides. The QD ζ-potential correlated with the ratio of modifiers. Azides on the QD surface could be easily transformed under mild conditions into primary amines using tris(2-carboxyethyl)phosphine as a reducing agent. The applicability of surface-functionalized QDs for bioconjugation was demonstrated via conjugation to fluorescent-dye-labeled bovine serum albumin.
Amine- and carboxyl-terminal self-assembled monolayers (SAMs) have been used frequently for controlling the adsorption of biomolecules. p-Aminophenol (p-AP) redox cycling by reducing reagents have ...been used to improve the sensitivity for signal amplification detection. However, most of the reported reducing reagents are unsuitable for gold electrodes due to the high background current caused by the oxidation reaction of the reducing reagents on highly electrocatalytic gold electrodes. Thiols and tris(2-carboxyethyl)phosphine (TCEP) are well-known reagents for reducing quinone. In this work, we evaluated the performances of p-AP redox cycling by TCEP and thiols on cysteamine- and 3-mercaptopropionic acid (MPA)-terminal gold electrodes. The results indicated that TCEP is more suitable for p-AP redox cycling on the modified gold electrode because of its negligible background current and fast chemical reaction with the oxidized p-AP. The work will be valuable for the development of affinity biosensors with p-AP redox cycling on gold substrates.
► We describe a method for metabolic labeling of palmitoylated Wnt and Hh. ► We use w-alkynyl palmitic acid and Click chemistry. ► The labeling does not interfere with Wnt or Hh signaling. ► ...Palmitoylated Wnt and Hh proteins are found in cellular membrane fractions. ► The method is useful for validating inhibitors of Wnt and Hh acyltransferases.
Palmitoylation of the Wnt and Hedgehog proteins is critical for maintaining their physiological functions. To date, there are no reported studies that characterize the cellular distribution of the palmitoylated forms of these proteins. Here, we describe the subcellular localization of palmitoylated Wnt and Sonic Hedgehog by using a highly sensitive and non-radioactive labeling method that utilizes alkynyl palmitic acid. We show that palmitoylated Wnt and Sonic Hedgehog localize to cellular membrane fractions only, highlighting a role for palmitoylation in the membrane association of these proteins. The method described herein has the utility to validate inhibitors of Wnt and Hedgehog acyltransferases in drug discovery, and enables further investigations of the role of palmitoylation in the secretion and signaling of these proteins.
•A thermodynamic cycle for cooperativity during translation inhibition is proposed.•Affinity of 4E-BP1 to eIF4E increases markedly upon binding to mRNA 5′ cap.•4E-BP1 flanking regions play some role ...in binding to eIF4E/cap but not to apo-eIF4E.•4E-BP1 stabilizes the eIF4E conformation capable of binding the cap.•4E-BP1 facilitates dissociation of eIF4E from the mRNA 5′ cap.
Initiation is the rate-limiting step during mRNA 5′ cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.
4E-BP1 and eIF4Ebind by cosedimentation in solution (View interaction)
Trivalent phosphines have been used for disulfide reduction for many years (1, 2). Tris2-carboxyethylphosphine (TCEP) has the added advantages of water solubility, broad pH stability, and minimal ...reactivity toward other functional groups found in proteins. In contrast to reducing agents such as dithiothreitol, TCEP does not contain thiols, and thus its removal is not required before subsequent modification of reduced proteins and peptides. However, TCEP can react with maleimidyl, iodoacetic acid and iodoacetamide compounds commonly used for protein and peptide modification (3, 4). For this reason, agarose gel-immobilized TCEP has been commercially developed (e.g., Pierce, G-Biosciences) to allow immediate use of TCEP-reduced proteins and peptides without requiring gel filtration or dialysis.
► We have structurally and functionally characterized Helicobacter pylori DsbG. ► The monomeric and dimeric structures of H. pylori DsbG show significant differences compared to E. coli DsbG. ► H. ...pylori DsbG shows a reductase activity against HP0518, a putative l,d-transpeptidase in H. pylori.
Dsb proteins play important roles in bacterial pathogenicity. To better understand the role of Dsb proteins in Helicobacter pylori, we have structurally and functionally characterized H. pylori DsbG (HP0231). The monomer consists of two domains connected by a helical linker. Two monomers associate to form a V-shaped dimer. The monomeric and dimeric structures of H. pylori DsbG show significant differences compared to Escherichia coli DsbG. Two polyethylene glycol molecules are bound in the cleft of the V-shaped dimer, suggesting a possible role as a chaperone. Furthermore, we show that H. pylori DsbG functions as a reductase against HP0518, a putative l,d-transpeptidase with a catalytic cysteine residue.
DsbG and DsbGbind by X-ray crystallography (View interaction).
Thiol-disulfide exchange is a key posttranslational modification, determining the folding process of intra- and inter-protein structures. Thiols can be detected by colorimetric reagents, which are ...stoichiometrically reduced by free thiols, and by fluorescent adducts, showing fluorescence only after thioester formation. We adapted a simple three-step method for detection of disulfide bonds in proteins. After irreversible blocking of protein thiols, disulfide bonds are reduced, followed by the detection of thiols. The approach presented here provides an economical procedure that can be used to obtain a global overview of the thiol-disulfide status of proteins in plants. This method allows the detection of modifications in samples on a gel and can be used for semi-quantitative analysis.
C(2) domains are well characterized as Ca(2+)/phospholipid-binding modules, but little is known about how they mediate protein-protein interactions. In neurons, a Munc13-1 C(2)A-domain/RIM ...zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13-1 C(2)A domain homodimerizes, and that homodimerization competes with Munc13-1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13-1 C(2)A-domain homodimer and the Munc13-1 C(2)A-domain/RIM ZF heterodimer at 1.44 A and 1.78 A resolution, respectively. The C(2)A domain adopts a beta-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded beta-barrel. In contrast, heterodimerization involves the bottom tip of the C(2)A-domain beta-sandwich and a C-terminal alpha-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13-1 homodimer-Munc13-1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C(2) domains as protein-protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes.
The paper reports modification and characterization of wool fabrics achieved through thiol-epoxy click chemistry. A pretreatment with tris (2-carboxyethyl) phosphine (TCEP) as an effective reducing ...agent was carried out to produce thiol groups on wool surface. Glycidyl trimethyl ammonium chloride (GTAC) was later covalently bonded with wool fibers via thiol-epoxy reaction. The reaction was confirmed by SEM, FTIR, Raman and TG analysis. Antibacterial activity, antistatic property, hydrophilicity and dyeability of treated wool fabric were assessed. The results demonstrated that TCEP-GTAC treatment can endow wool fabric good antibacterial and antistatic properties as well as improved hydrophilicity. Tensile strength studies indicated fiber strength loss of ~12 % on modification.
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