The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP

J Biotechnol. 2004 Aug 26;112(1-2):151-64. doi: 10.1016/j.jbiotec.2004.04.030.


Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 [Mol. Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group Ia, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg(2+) on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg(2+). Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Computer Simulation
  • Fluorescence Resonance Energy Transfer / methods*
  • GTP Phosphohydrolases / chemistry*
  • GTP-Binding Proteins / chemistry
  • Guanosine Triphosphate / chemistry*
  • Models, Chemical
  • Models, Molecular*
  • Molecular Sequence Data
  • Multiprotein Complexes / analysis
  • Multiprotein Complexes / chemistry
  • Plant Proteins / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Mapping / methods
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Surface Plasmon Resonance / methods*
  • rac1 GTP-Binding Protein / chemistry*


  • Multiprotein Complexes
  • Plant Proteins
  • Guanosine Triphosphate
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • rac1 GTP-Binding Protein