Use of a Fluorescent ATP Analog to Probe the Allosteric Conformational Change in the Active Site of the Protein Kinase PDK1

  • Valerie Hindie
  • Laura A. Lopez-Garcia
  • Ricardo M. BiondiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 928)


There is growing interest in exploring allosteric sites on proteins for drug discovery. At the center of the regulation of many protein kinases from the AGC family there is an allosteric site termed “PIF-pocket.” The regulated binding of a C-terminal region of the kinase to the PIF-pocket, within the small lobe of the catalytic core, modulates the activity of AGC kinases. Small compounds that bind to the PIF-pocket can mimic its physiological mechanism of regulation and modulate the kinase activity in vitro, e.g., small compounds can activate the phosphoinositide-dependent protein kinase 1 (PDK1). Compounds binding to an allosteric site on a protein kinase may produce conformational changes at the ATP-binding site within the active site of the kinase domain. We here describe a fluorescent method using the ATP analog TNP-ATP that allows evaluating the allosteric conformational changes at the ATP-binding site of PDK1 triggered by small compounds binding to the PIF-pocket.

Key words

PDK1 AGC protein kinase Conformational change Allosteric site ATP-binding site 


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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Valerie Hindie
    • 1
  • Laura A. Lopez-Garcia
    • 1
  • Ricardo M. Biondi
    • 1
    Email author
  1. 1.Department of Internal Medicine I, Research Group PhosphoSitesUniversitätsklinikum FrankfurtFrankfurtGermany

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