Understanding the topology adopted by individual G-quadruplex (GQ)-forming sequences in vivo and targeting a specific GQ motif among others in the genome will have a profound impact on GQ-directed therapeutic strategies. However, this remains a major challenge as most of the tools poorly distinguish different GQ conformations and are not suitable for both cell-free and in-cell analysis. Here, we describe an innovative probe design to investigate GQ conformations and recognition in both cell-free and native cellular environments by using a conformation-sensitive dual-app nucleoside analogue probe. The nucleoside probe, derived by conjugating fluorobenzofuran at the 5-position of 2'-deoxyuridine, is composed of a microenvironment-sensitive fluorophore and an in-cell NMR compatible F label. This noninvasive nucleoside, incorporated into the human telomeric DNA oligonucleotide repeat, serves as a common probe to distinguish different GQ topologies and quantify topology-specific binding of ligands by fluorescence and NMR techniques. Importantly, unique signatures displayed by theF-labeled nucleoside for different GQs enabled a systematic study in Xenopus laevis oocytes to provide new structural insights into the GQ topologies adopted by human telomeric overhang in cells, which so far has remained unclear. Studies using synthetic cell models, immunostaining on fixed cells, and crystallization conditions suggest that parallel GQ is the preferred conformation of telomeric DNA repeat. However, our findings using the dual-app probe clearly indicate that multiple structures including hybrid-type parallel-antiparallel and parallel GQs are formed in the cellular environment. Taken together, our findings open new experimental strategies to investigate topology, recognition, and therapeutic potential of individual GQ-forming motifs in a biologically relevant context.

Fever is a conserved and prominent response to infection. Yet, the issue of how CD4 T cell responses are modulated if they occur at fever temperatures remains poorly addressed. We have examined the priming of naive CD4 T cells in vitro at fever temperatures, and we report notable fever-mediated modulation of their cytokine commitment. When naive CD4 T cells were primed by plate-bound anti-CD3 and anti-CD28 monoclonal antibodies at moderate fever temperature (39 °C), they enhanced commitment to IL4/5/13 (Th2) and away from IFNg (Th1). This was accompanied by up-regulation of the Th2-relevant transcription factor GATA3 and reduction in the Th1-relevant transcription factor Tbet. Fever sensing by CD4 T cells involved transient receptor potential vanilloid cation channels (TRPVs) since TRPV1/TRPV4 antagonism blocked the febrile Th2 switch, while TRPV1 agonists mediated a Th2 switch at 37 °C. The febrile Th2 switch was IL4 independent, but a γ-secretase inhibitor abrogated it, and it was not found in Notch1-null CD4 T cells, identifying the Notch pathway as a major mediator. However, when naive CD4 T cells were primed via antigen and dendritic cells (DCs) at fever temperatures, the Th2 switch was abrogated via increased production of IL12 from DCs at fever temperatures. Thus, immune cells directly sense fever temperatures with likely complex physiological consequences.

The roles of surrounding membrane lipids in the functions of transmembrane and peripheral membrane proteins are largely unknown. Herein, we utilize the recently reported structures of the TRPV1 ion channel protein bound to its potent protein agonist, the double-knot toxin (DkTx), as a model system to investigate the roles of toxin-lipid interfaces in TRPV1 activation by characterizing a series of DkTx variants electrophysiologically. Together with membrane partitioning experiments, these studies reveal that toxin-lipid interfaces play an overwhelmingly dominant role in channel activation as compared to lipid-devoid toxin-channel interfaces. Additionally, we find that whereas the membrane interfaces formed by one of the knots of the toxin endow it with its low channel-dissociation rate, those formed by other knot contribute primarily to its potency. These studies establish that protein-lipid interfaces play nuanced yet profound roles in the function of protein-protein complexes within membranes.

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.