Watching C 'n' N: Hydrazones and oximes are common conjugates but labile to hydrolysis. The hydrolytic stabilities of isostructural hydrazones one oxime were determined at pD 5.0–9.0. rate constant for the acid-catalyzed hydrolysis was nearly 103-fold lower than those simple hydrazones, a trialkylhydrazonium ion (formed after condensation) even more stable oxime. Supporting information this article is available on WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z802651_s.pdf or from...

Bioconjugation is a burgeoning field of research. Novel methods for the mild and site-specific derivatization proteins, DNA, RNA, carbohydrates have been developed applications such as ligand discovery, disease diagnosis, high-throughput screening. These powerful owe their existence to discovery chemoselective reactions that enable bioconjugation under physiological conditions-a tremendous achievement modern organic chemistry. Here, we review recent advances in Additionally, discuss...

O toppt N, nicht aber N+: Hydrazone und Oxime sind gängige, doch leicht hydrolysierbare Verbindungen. Die Hydrolysestabilitäten isostruktureller von einem Oxim wurden bei pD 5.0–9.0 untersucht. Geschwindigkeitskonstante der säurekatalysierten Hydrolyse war beim fast 103-mal kleiner als einfachen Hydrazonen, ein Trialkylhydrazoniumion (durch Kondensation gebildet) noch stabiler das Oxim. Supporting information for this article is available on the WWW under...

Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve double-knot toxin (DkTx), a tarantula that activates heat-activated TRPV1 channel. We also provide improved structures with without bound, investigate interactions DkTx channel membranes. find binds to outer edge external pore in counterclockwise configuration, using limited protein-protein interface inserting hydrophobic residues into bilayer. show partitions naturally membranes, two...

Voltage‐gated ion channels (VGICs) are allosterically modulated by glycosaminoglycan proteoglycans and sialic acid glycans. However, the structural diversity heterogeneity of these biomolecules pose significant challenges to precisely delineate their underlying structure‐activity relationships. Herein, we demonstrate how heparan sulfate (HS) synthetic glycans appended on amphiphilic glycopeptide backbone influence cell membrane persistence modulate gating Kv2.1 channel. Utilizing a panel...

Protein microarrays are playing an increasingly important role in the discovery and characterization of protein−ligand interactions. The uniform orientation conferred by site-specific immobilization is a demonstrable advantage using such microarrays. Here, we report on general strategy for fabricating gold surfaces displaying protein orientation. An azido group was installed at C-terminus model protein, bovine pancreatic ribonuclease, method expressed ligation synthetic bifunctional reagent....

Here, we report the discovery of a novel anticonvulsant drug with molecular organization based on unique scaffold rufinamide, an anti-epileptic compound used in clinical setting to treat severe epilepsy disorders such as Lennox-Gastaut syndrome. Although accumulating evidence supports working mechanism through voltage-gated sodium (Nav) channels, found that clinically relevant rufinamide concentration inhibits human (h)Nav1.1 activation, distinct among anticonvulsants and feature worth...

Significance Since fever is frequently a response to infection, immune responses likely occur commonly at temperatures. Are temperatures detected by cells, what molecular pathways are involved in this sensing, and the functional consequences? Our data show that CD4 T lymphocytes detect moderate temperature. A TRPV channel-mediated pathway involving Notch activation leads Th2 skewing of effector cell response. However, antigen presenting dendritic cells stimulate turn, temperature overproduce...

The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is ability modify a target protein at specific site with functional group orthogonal reactivity. Here, we achieve that by exploiting intrinsic electrophilicity thioester intermediate formed during intein-mediated splicing. Detailed kinetic analyses reaction nitrogen nucleophiles chromogenic small-molecule revealed alpha-hydrazino acetyl was optimal nucleophile...

Paucity of efficient probes and small molecule ligands that can distinguish different G-quadruplex (GQ) topologies poses challenges not only in understanding their basic structure but also targeting an individual GQ form from others. Alternatively, G-rich sequences harbour unique chimeric structural motifs (e.g., GQ-duplex or GQ-hairpin junctions) are perceived as new therapeutic hotspots. In this context, the epidermal growth factor receptor (EGFR) gene, implicated many cancers, contains a...

Understanding the structure and recognition of highly conserved regulatory segments integrated viral DNA genome that forms unique topologies can greatly aid in devising novel therapeutic strategies to counter chronic infections. In this study, we configured a probe system using environment-sensitive nucleoside analogs, 5-fluoro-2'-deoxyuridine (FdU) 5-fluorobenzofuran-2'-deoxyuridine (FBFdU), investigate structural polymorphism HIV-1 long terminal repeat (LTR) G-quadruplexes (GQs) by...

The molecular mechanisms underlying the activation of Transient Receptor Potential (TRP) ion channels are poorly understood when compared to those voltage-activated potassium (Kv) channels. architectural and pharmacological similarities between members these two families suggest that their structure-function relationships may have common features. We explored this hypothesis by replacing previously identified domains critical structural motifs membrane-spanning portions Kv2.1 with...

A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of tarantula Ornithoctonus huwena and found to stably activate TRPV1 channels by targeting outer pore domain. DkTx has been shown consist two inhibitory cysteine-knot (ICK) motifs, referred as K1 K2, each containing six cysteine residues. Beyond this initial characterization, however, structural functional details about remains elusive in large part due lack a high yielding methodology for synthesis...

Guanidine and its alkyl analogs stimulate the neuromuscular junction presynaptically by inhibiting voltage-gated potassium (Kv) channels, leading to enhanced release of acetylcholine in synaptic cleft. This stimulatory effect guanidine underlies use therapy for diseases myasthenic syndrome Lambert-Eaton botulism. The therapeutic is limited, however, because side effects that accompany administration. Therefore, design with improved indices desirable. Progress toward this goal hindered lack...

Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we extended these methods to release and modify intein fusion proteins on yeast surface, thereby eliminating need for expression purification. To this end, sought simultaneously surface-displayed selectively conjugate with functionalities compatible EPL click chemistry. Single-chain antibodies (scFv) green...

Rapid, catalyst-free and reversible bioconjugation in mammalian cells.

Choline is an essential nutrient for mammalian cells. Our understanding of the cellular functions choline and its metabolites, independent their roles as lipid metabolism intermediates, remains limited. In addition to fundamental physiology, this knowledge has implications cancer biology because elevated metabolite levels are a hallmark cancer. Here, we establish metabolite-interacting proteome by utilizing photocrosslinkable probe. To design probe, performed metabolic labeling experiments...

The roles of surrounding membrane lipids in the functions transmembrane and peripheral proteins are largely unknown. Herein, we utilize recently reported structures TRPV1 ion channel protein bound to its potent agonist, double-knot toxin (DkTx), as a model system investigate toxin–lipid interfaces activation by characterizing series DkTx variants electrophysiologically. Together with partitioning experiments, these studies reveal that play an overwhelmingly dominant role compared...

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these for target channels enables them serve as powerful tools biology. Toxins labeled with fluorescent dyes are employed the cellular imaging also studying toxin-channel toxin-membrane interactions. Several cysteine-rich, rendering production properly folded fluorescently technically challenging. Herein, we evaluate a variety site-specific protein...

The spider venom protein, double-knot toxin (DkTx), partitions into the cellular membrane and binds bivalently to pain-sensing ion channel, TRPV1, triggering long-lasting channel activation. In contrast, its monovalent single knots partition poorly invoke rapidly reversible TRPV1 To discern contributions of bivalency affinity DkTx sustained mode action, here, we developed diverse variants including those containing truncated linkers between individual knots, precluding bivalent binding....