Site-Specific Fluorescent Labeling of the Cysteine-Rich Toxin, DkTx, for TRPV1 Ion Channel Imaging and Membrane Binding Studies.

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover, N-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches, C-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed in Xenopus laevis oocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.

Tertiary-Butylbenzene Functionalization as a Strategy for ß-Sheet Polypeptides.

ß-Sheet forming polypeptides are one of the least explored synthetic systems due to their uncontrolled precipitation in the ring-opening polymerization (ROP) synthetic methodology. Here, a new t-butylbenzene functionalization approach is introduced to overcome this limitation by sterically controlling the propagating polymer chains, and homogeneous polymerization with good control over chain growth was accomplished. New bulky N-carboxyanhydride monomers were designed having t-butylbenzene pendant by multistep organic synthesis, and N-heterocyclic carbene was explored as a catalyst to make high-molecular-weight and narrow polydisperse soluble polypeptides. This ROP process was successfully demonstrated for two ß-sheet forming polypeptides such as poly(l-serine) and poly(l-cysteine). These new t-butylbenzene-functionalized polypeptides were found to be readily soluble in tetrahydrofuran, chloroform, and so forth, and they were produced in high molecular weights having Mn = 32 kDa with dispersity D̵ ≤ 1.3. ROP kinetics were studied by real-time Fourier transform infrared and 1H NMR to determine the actual content of the secondary structures in the propagating chains. These studies established that the α-helical conformational front in the propagation chain was speeding up the polymerization kinetics with good degree of control in the ROP process. Reversible-conformational transitions in the post-polymerization deprotection were found to restore the ß-sheet secondary structures in poly(l-serine)s. The newly developed t-butylbenzene-substituted steric-hindrance approach is valuable in yielding soluble polymers, and this approach could be useful for exploring new polypeptide architectures for long-term impact.

Rapid and reversible hydrazone bioconjugation in cells without the use of extraneous catalysts.

The amenability of hydrazone linkages to disassemble via either hydrolysis in mildly acidic aqueous solutions or transimination upon treatment with amine nucleophiles renders them extremely attractive for applications in chemical biology, drug delivery and materials science. Unfortunately, however, the use of hydrazones is hampered by the extremely slow intrinsic rates of their formation from their hydrazine and carbonyl precursors. Consequently, hydrazone formation is typically performed in the presence of a large excess of cytotoxic aniline-based nucleophilic catalysts, rendering hydrazones unsuitable for biological applications that entail their formation in cells. Herein, we report a hydrazine scaffold-o-amino benzyl hydrazine-that rapidly forms hydrazones via intramolecular nucleophilic catalysis, thereby obviating the use of extraneous catalysts. We demonstrate the use of this scaffold for rapid and reversible peptide and protein hydrazone bioconjugation and also for reversible fluorescent labeling of sialylated glycoproteins and choline lipids in mammalian cells.