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Characterizing Plexin GTPase Interactions Using Gel Filtration, Surface Plasmon Resonance Spectrometry, and Isothermal Titration Calorimetry

  • Jeannine Muller-Greven
  • SoonJeung Kim
  • Prasanta K. Hota
  • Yufeng Tong
  • Susmita Borthakur
  • Matthias BuckEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1493)

Abstract

Plexins are unique, as they are the first example of a transmembrane receptor that interacts directly with small GTPases, a family of proteins that are essential for cell motility and proliferation/survival. We and other laboratories have determined the structure of the Rho GTPase-binding domain (RBD) of several plexins and also of the entire intracellular region of plexin-B1. Structures of plexin complexes with Rho GTPases, Rac1 and Rnd1, and a structure with a Ras GTPase, Rap1b, have also been solved. The relationship between plexin-Rho and plexin-Ras interactions is still unclear and in vitro biophysical experiments that characterize the protein interactions of purified components play an important role in advancing our understanding of the molecular mechanisms that underlie the function of plexin. This chapter describes the use of gel filtration (also known as size-exclusion chromatography or SEC), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) in studies of plexin—small GTPase interactions with plexin-B1:Rac1 as an example. Together with other assays and manipulations (e.g., by mutagenesis or protein domain truncation/deletion), these in vitro measurements provide an important reference for the role and extent of the interactions.

Key words

Protein binding affinity Protein complex formation Rho GTPases Rac1 Size-exclusion chromatography [SEC] Surface plasmon resonance spectrometry [SPR] Isothermal titration calorimetry [ITC] 

Notes

Acknowledgements

We thank Drs. Shufen Cao, Liqun Zhang, and other members of the Buck lab for insightful discussion. The work of M.B. was supported by the NIH grants R01GM92851 and R01GM73071.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Jeannine Muller-Greven
    • 1
  • SoonJeung Kim
    • 1
  • Prasanta K. Hota
    • 1
  • Yufeng Tong
    • 2
  • Susmita Borthakur
    • 1
  • Matthias Buck
    • 1
    • 3
    • 4
    • 5
    • 6
    Email author
  1. 1.Department of Physiology and BiophysicsCase Western Reserve University, School of MedicineClevelandUSA
  2. 2.Structural Genomics Consortium, Department of Pharmacology and ToxicologyUniversity of TorontoTorontoCanada
  3. 3.Department of NeurosciencesCase Western Reserve University, School of MedicineClevelandUSA
  4. 4.Department of PharmacologyCase Western Reserve University, School of MedicineClevelandUSA
  5. 5.Case Comprehensive Cancer CenterCase Western Reserve University, School of MedicineClevelandUSA
  6. 6.Center for Proteomics and BioinformaticsCase Western Reserve University, School of MedicineClevelandUSA

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