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

Methods Mol Biol. 2017:1493:89-105. doi: 10.1007/978-1-4939-6448-2_6.

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.

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

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Calorimetry / methods*
  • Cell Adhesion Molecules / metabolism*
  • Chromatography, Gel / methods*
  • Gene Expression
  • Nerve Tissue Proteins / metabolism*
  • Surface Plasmon Resonance / methods*
  • Thermodynamics
  • rho GTP-Binding Proteins / metabolism*

Substances

  • Cell Adhesion Molecules
  • Nerve Tissue Proteins
  • plexin
  • rho GTP-Binding Proteins