Binding Affinity Measurement of Nuclear Export Signal Peptides to Their Exporter CRM1

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

CRM1 recognizes hundreds to thousands of protein cargoes by binding to the eight to fifteen residue-long nuclear export signals (NESs) within their polypeptide chains. Various assays to measure the binding affinity of NESs for CRM1 have been developed. CRM1 binds to NESs with a wide range of binding affinities, with dissociation constants that span from low nanomolar to tens of micromolar. An optimized binding affinity assay with improved throughput was recently developed to measure binding affinities of NES peptides for CRM1 in the presence of excess RanGTP. The assay can measure affinities, with multiple replicates, for up to seven different NES peptides per screening plate. Here, we present a protocol for the purification of the necessary proteins and for measuring CRM1-NES binding affinities.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages245-256
Number of pages12
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2502
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Binding affinity
  • CRM1
  • Fluorescence polarization
  • NES
  • Nuclear export signals
  • XPO1

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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