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.

[1]  G. Agrawal,et al.  Small GTPase ‘Rop’: molecular switch for plant defense responses , 2003, FEBS letters.

[2]  Zhenbiao Yang,et al.  Arabidopsis Rho-related GTPases: differential gene expression in pollen and polar localization in fission yeast. , 1998, Plant physiology.

[3]  K. Shimamoto,et al.  The small GTP-binding protein rac is a regulator of cell death in plants. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Myszka,et al.  Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. , 1997, Current opinion in biotechnology.

[5]  A. Hall,et al.  The effect of Mg2+ on the guanine nucleotide exchange rate of p21N-ras. , 1986, The Journal of biological chemistry.

[6]  S F Altschul,et al.  Protein database searches for multiple alignments. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[7]  E. Kretschmann,et al.  Notizen: Radiative Decay of Non Radiative Surface Plasmons Excited by Light , 1968 .

[8]  R. Karlsson,et al.  Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. , 1991, BioTechniques.

[9]  I. Schlichting,et al.  Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras. , 1993, The Journal of biological chemistry.

[10]  R. Hückelhoven,et al.  A Small GTP-Binding Host Protein Is Required for Entry of Powdery Mildew Fungus into Epidermal Cells of Barley1 , 2002, Plant Physiology.

[11]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[12]  P. Wojtaszek.  Oxidative burst: an early plant response to pathogen infection. , 1997, The Biochemical journal.

[13]  Y. Yuasa,et al.  Optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus. , 1993, Analytical biochemistry.

[14]  R. Dixon,et al.  A Plant Homolog of the Neutrophil NADPH Oxidase gp91phox Subunit Gene Encodes a Plasma Membrane Protein with Ca2+ Binding Motifs , 1998, Plant Cell.

[15]  Manuel C. Peitsch,et al.  SWISS-MODEL: an automated protein homology-modeling server , 2003, Nucleic Acids Res..

[16]  C. Der,et al.  Biological and biochemical properties of human ras H genes mutated at codon 61 , 1986, Cell.

[17]  Frank McCormick,et al.  The GTPase superfamily: a conserved switch for diverse cell functions , 1990, Nature.

[18]  B. Liedberg,et al.  Surface plasmon resonance for gas detection and biosensing , 1983 .

[19]  K Furukawa,et al.  Kinetic analysis of DNA binding by the c‐Myb DNA‐binding domain using surface plasmon resonance , 1999, FEBS letters.

[20]  M. Nakafuku,et al.  Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants. , 1988, Biochimica et biophysica acta.

[21]  R. Karlsson,et al.  Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis. , 1992, Journal of chromatography.

[22]  M. Zerial,et al.  Kinetics of Interaction of Rab5 and Rab7 with Nucleotides and Magnesium Ions* , 1996, The Journal of Biological Chemistry.

[23]  Frank McCormick,et al.  The GTPase superfamily: conserved structure and molecular mechanism , 1991, Nature.

[24]  T. Hiratsuka.  New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. , 1983, Biochimica et biophysica acta.

[25]  T. Boller.  CHEMOPERCEPTION OF MICROBIAL SIGNALS IN PLANT CELLS , 1995 .

[26]  P. Low,et al.  The oxidative burst in plant defense: Function and signal transduction , 1996 .

[27]  J Otlewski,et al.  Structure of small G proteins and their regulators. , 2001, Acta biochimica Polonica.

[28]  M C Peitsch,et al.  Protein structure computing in the genomic era. , 2000, Research in microbiology.

[29]  A. Plückthun,et al.  Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. I. GroEL recognizes the signal sequences of beta-lactamase precursor. , 1994, Journal of molecular biology.

[30]  R. Neubig,et al.  Fluorescent guanine nucleotide analogs and G protein activation. , 1994, The Journal of biological chemistry.

[31]  Zhenbiao Yang.  Small GTPases: versatile signaling switches in plants. , 2002, The Plant cell.

[32]  L. Henderson,et al.  NADPH oxidase of neutrophils. , 1996, Biochimica et biophysica acta.

[33]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[34]  A. Jesaitis,et al.  Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. , 1993, The Journal of biological chemistry.

[35]  T. Brembu,et al.  Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana , 1997, Plant Molecular Biology.

[36]  I. Schlichting,et al.  Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli. , 1990, The Journal of biological chemistry.

[37]  J. Erickson,et al.  Real‐time DNA binding measurements of the ETSl recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms , 1994, Protein science : a publication of the Protein Society.

[38]  M. Hirshberg,et al.  The crystal structure of human rac1, a member of the rho-family complexed with a GTP analogue , 1997, Nature Structural Biology.

[39]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[40]  J. Chernoff,et al.  Plant GTPases: the Rhos in bloom. , 2000, Trends in cell biology.

[41]  I. Rönnberg,et al.  Flow-injection ellipsometry ― an in situ method for the study of biomolecular adsorption and interaction at solid surfaces , 1985 .

[42]  K. Niehaus,et al.  Transgenic tobacco plants that express an antisense construct derived from a Medicago sativa cDNA encoding a Rac-related small GTP-binding protein fail to develop necrotic lesions upon elicitor infiltration , 2000, Molecular and General Genetics MGG.

[43]  E T Young,et al.  An accessory DNA binding motif in the zinc finger protein Adr1 assists stable binding to DNA and can be replaced by a third finger. , 2000, Biochemistry.

[44]  M. Hämäläinen,et al.  Kinetic analysis of the interaction between HIV-1 protease and inhibitors using optical biosensor technology. , 2000, Analytical biochemistry.

[45]  I. Macara,et al.  Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A. , 1992, The Journal of biological chemistry.

[46]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[47]  T. Sekiya,et al.  Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[48]  R. Clegg.  Fluorescence resonance energy transfer. , 2020, Current Opinion in Biotechnology.