title: Characterizing Plexin GTPase Interactions Using Gel Filtration, Surface Plasmon Resonance Spectrometry, and Isothermal Titration Calorimetry.
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.
authors: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH, 44106, USA.
authors: Jeannine
authors: J
authors: Muller-Greven
authors: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH, 44106, USA.
authors: SoonJeung
authors: S
authors: Kim
authors: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH, 44106, USA.
authors: Prasanta K
authors: PK
authors: Hota
authors: Structural Genomics Consortium, Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, M5G 1L7, Canada.
authors: Yufeng
authors: Y
authors: Tong
authors: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH, 44106, USA.
authors: Susmita
authors: S
authors: Borthakur
authors: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH, 44106, USA. Matthias.Buck@case.edu.|Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, OH, 44106, USA. Matthias.Buck@case.edu.|Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, OH, 44106, USA. Matthias.Buck@case.edu.|Case Comprehensive Cancer Center, Case Western Reserve University, School of Medicine, Cleveland, OH, 44106, USA. Matthias.Buck@case.edu.|Center for Proteomics and Bioinformatics, Case Western Reserve University, School of Medicine, Cleveland, OH, 44106, USA. Matthias.Buck@case.edu.
authors: Matthias
authors: M
authors: Buck
mesh_terms: D002151:Calorimetry / Q000379:methods*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods*; D015870:Gene Expression; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013816:Thermodynamics; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; D009419:Nerve Tissue Proteins; C091529:plexin; D020741:rho GTP-Binding Proteins
keywords: Isothermal titration calorimetry [ITC]; Protein binding affinity; Protein complex formation; Rac1; Rho GTPases; Size-exclusion chromatography [SEC]; Surface plasmon resonance spectrometry [SPR]