Jeet Kalia

Hydrolytic Stability of Hydrazones and Oximes

Hydrazones and oximes are common conjugates, but are labile to hydrolysis. The hydrolytic stability of isostructural hydrazones and an oxime have been determined at pD 5.0–9.0. The hydrolysis of each adduct was catalyzed by acid. Rate constants for oxime hydrolysis were nearly 103-fold lower than those for simple hydrazones; a trialkylhydrazonium ion (formed after condensation) was even more stable than the oxime. The data suggest a general mechanism for conjugate hydrolysis.

Advances in Bioconjugation.

Bioconjugation is a burgeoning field of research. Novel methods for the mild and site-specific derivatization of proteins, DNA, RNA, and carbohydrates have been developed for applications such as ligand discovery, disease diagnosis, and high-throughput screening. These powerful methods owe their existence to the discovery of chemoselective reactions that enable bioconjugation under physiological conditions-a tremendous achievement of modern organic chemistry. Here, we review recent advances in bioconjugation chemistry. Additionally, we discuss the stability of bioconjugation linkages-an important but often overlooked aspect of the field. We anticipate that this information will help investigators choose optimal linkages for their applications. Moreover, we hope that the noted limitations of existing bioconjugation methods will provide inspiration to modern organic chemists.

From foe to friend: using animal toxins to investigate ion channel function.

Ion channels are vital contributors to cellular communication in a wide range of organisms, a distinct feature that renders this ubiquitous family of membrane-spanning proteins a prime target for toxins found in animal venom. For many years, the unique properties of these naturally occurring molecules have enabled researchers to probe the structural and functional features of ion channels and to define their physiological roles in normal and diseased tissues. To illustrate their considerable impact on the ion channel field, this review will highlight fundamental insights into toxin-channel interactions and recently developed toxin screening methods and practical applications of engineered toxins.

Reactivity of intein thioesters: appending a functional group to a protein.

The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein‐mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small‐molecule thioester revealed that the α‐hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an α‐hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3‐dipolar azide–alkyne cycloaddition. Thus, the mechanism of intein‐mediated protein splicing provides the means to install a useful functional group at a specific site—the C terminus—of virtually any protein.

Exploring structure-function relationships between TRP and Kv channels

The molecular mechanisms underlying the activation of Transient Receptor Potential (TRP) ion channels are poorly understood when compared to those of the voltage-activated potassium (Kv) channels. The architectural and pharmacological similarities between the members of these two families of channels suggest that their structure-function relationships may have common features. We explored this hypothesis by replacing previously identified domains and critical structural motifs of the membrane-spanning portions of Kv2.1 with corresponding regions of two TRP channels, TRPM8 and TRPV1. Our results show that the S3b-S4 paddle motif of Kv2.1, but not other domains, can be replaced by the analogous regions of both TRP channels without abolishing voltage-activation. In contrast, replacement of portions of TRP channels with those of Kv2.1 consistently yielded non-functional channels. Taken together, these results suggest that most structural elements within TRP channels and Kv channels are not sufficiently related to allow for the creation of hybrid channels.

Elucidating the molecular basis of action of a classic drug: guanidine compounds as inhibitors of voltage-gated potassium channels.

Guanidine and its alkyl analogs stimulate the neuromuscular junction presynaptically by inhibiting voltage-gated potassium (Kv) channels, leading to enhanced release of acetylcholine in the synaptic cleft. This stimulatory effect of guanidine underlies its use in the therapy for the neuromuscular diseases myasthenic syndrome of Lambert-Eaton and botulism. The therapeutic use of guanidine is limited, however, because of side effects that accompany its administration. Therefore, the design of guanidine analogs with improved therapeutic indices is desirable. Progress toward this goal is hindered by the lack of knowledge of the mechanism by which these molecules inhibit Kv channels. Here we examine an array of possible mechanisms, including charge screening, disruption of the protein-lipid interfaces, direct interaction with the voltage sensors, and pore-binding. Our results demonstrate that guanidines bind within the intracellular pore of the channel and perturb a hydrophobic subunit interface to stabilize a closed state of the channel. This mechanism provides a foundation for the design of guanidine analogs for the therapeutic intervention of neuromuscular diseases.

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide-alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.

Structural Characterization of Double-Knot Toxin, an Activator of TRPV1 Channels

Venom from poisonous organisms is a rich source of peptide toxins interacting with different ion channels proteins. These peptide toxins modulate ion channels by different mechanisms, and have been widely used as tools for investigating ion channel mechanisms. Double-knot toxin (DkTx) is a novel peptide toxin that activates TRPV1 channels, and contains two inhibitory cysteine knot (ICK) motifs, as its name suggests. Previous studies show that DkTx activates TRPV1 channels, and suggest that the avidity of the toxin (slow unbinding) arises from its bivalent nature. Here we use solid-phase peptide synthesis to individually produce the two knots of DkTx (K1 and K2), fold each in vitro, and find that they exhibit different binding affinities for the channel even though they share high sequence homology. As a first step toward understanding the structural and functional relationship of DkTx binding to TRPV1 channels, we determined solution structures of each knot in using NMR. The structures show that DkTx is composed of two notably amphipathic ICK motifs (each with two beta-strands) that are connected by a flexible linker, and that K2 has a larger hydrophobic surface compared to K1. In addition, the single conserved Trp residue in each knot show different orientations, with that in K1 exhibiting greater solvent exposure. Interestingly, using intrinsic Trp fluorescence, we observe strong partitioning of DkTx and K1, but see no evidence of membrane partitioning for K2. We also made a series of K1/K2 chimeras, and identified variant residues in two loops and the C-terminus that are responsible for the higher activity of K2. From these results we propose that membrane interactions are involved in the mechanisms of DkTx activation of TRPV1, and identify surfaces of the two knots that likely involved in binding to the channel.

The design principle of paddle motifs in voltage sensors

Voltage-activated ion channels contain S1–S4 domains that endow them with exquisite voltage sensitivity. X-ray crystal structures provided a major breakthrough in elucidating the mechanistic basis of voltage sensing, revealing a helix-turn-helix motif termed the voltage-sensor paddle. A study in this issue demonstrates that this motif exists in the open state of Kv channels when embedded in native biological membranes and puts forward new ideas about its functional role in the mechanism of voltage sensing.

1,9-Bis(2-pyridyl)-1,2,8,9-tetrathia-5-oxanonane

Disulfide crosslinking of proteins is typically performed by treating proteins bearing cysteine residues with small-molecule disulfide reagents. The process results in the formation of a mixed disulfide intermediate, which then reacts with the cysteine residue of another protein molecule to form the crosslinked product. This second step requires the intimate association of two large reactants. The ensuing steric hindrance can result in poor crosslinking yields. Here, we introduce a bis(disulfide) reagent in which activated disulfides are separated by linkers that can alleviate steric hindrance and thereby potentially increase the efficiency of crosslinking.