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.
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.
Brecht
Martina
M
Faculty of Biology, Genetics, University of Bielefeld, D-33501 Bielefeld, Germany.
Sewald
Katherina
K
Schiene
Karin
K
Keen
Gunnar
G
Fricke
Matthias
M
Sauer
Markus
M
Niehaus
Karsten
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance / Q000379:methods*; D020830:rac1 GTP-Binding Protein / Q000737:chemistry*
D046912:Multiprotein Complexes; D010940:Plant Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein