SWTY - A general peptide probe for homogeneous solution binding assay of 14-3-3 proteins

Meng Wu, Brian Coblitz, Sojin Shikano, Shunyou Long, Lisa M. Cockrell, Haian Fu, Min Li

Research output: Contribution to journalArticlepeer-review

Abstract

Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.

Original languageEnglish (US)
Pages (from-to)186-196
Number of pages11
JournalAnalytical biochemistry
Volume349
Issue number2
DOIs
StatePublished - Feb 15 2006

Keywords

  • 14-3-3
  • Binding assay
  • Dissociation constant
  • Fluorescence anisotropy
  • High-throughput screening
  • SWTY

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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