Abstract
Understanding protein mechanism has been greatly facilitated by recent advances in single particle cryo-EM, where multiple conformations of protein within a single dataset can be identified. However, experimentally ascertaining whether these states lie along the same pathway and deducing their temporal relationship within the pathway is not trivial. By using thermal titration methods and cryo-EM we have temporally resolved the nearly complete conformational trajectory of the vanilloid receptor TRPV1 by the potent vanilloid resiniferatoxin (RTx). Monitoring temperature-dependent population shifts within conformational ensembles of the RTx-bound TRPV1 revealed the order of conformational changes leading to channel opening, including critical intermediate states. RTx binding induces intracellular gate opening first, followed by selectivity filter dilation, then ultimately pore loop rearrangement to reach the final open state of TRPV1. Our analysis has revealed that this apparent conformational wave arises from the concerted, stepwise, additive conformational changes over many subdomains of TRPV1. Our understanding of the RTx-mediated long-range allostery of TRPV1 could help further therapeutic potential of RTx, which is a promising drug candidate for pain relief associated with advanced cancer or knee arthrithis. We believe our thermal titration approach to dissect conformational ensembles from cryo-EM can be extended to many other systems.
Published Version
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