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Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7

ABSTRACT: This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of Nav1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-2 and helix S3 of VSD2 in a chimeric channel system. Sample preparation of purified chimeric channels crosslinked with the photoprobes was based on the FASP protocol. Various digestion enzymes were evaluated in order to maximise the sequence coverage of this membrane protein. Finally, MeroX was employed in order to identify the sites of crosslinking. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on Nav channels.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Foteini Tzakoniati

LAB HEAD: Edward William Tate

PROVIDER: PXD015037 | Pride | 2019-11-18

REPOSITORIES: Pride

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