Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins.
New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003497:Cyclic N-Oxides / Q000138:chemical synthesis*; D004578:Electron Spin Resonance Spectroscopy; D019204:GTP-Binding Proteins / Q000032:analysis*; D006150:Guanine Nucleotides; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D013058:Mass Spectrometry; D010444:Peptide Elongation Factor Tu / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels
D003497:Cyclic N-Oxides; D006150:Guanine Nucleotides; D013113:Spin Labels; C050543:3'-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carbonyl)amino-3'-deoxyguanosine 5'-diphosphate; C050544:3'-(2,2,5,5-tetramethylpyrroline-1-oxyl-3-carbonyl)amino-3'-deoxyguanosine 5'-triphosphate; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D010444:Peptide Elongation Factor Tu