title: Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.
abstract: A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan;
authors.forename: Seigo
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan;
authors.forename: Kaori
authors.initials: K
authors.lastname: Masuhara
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan;
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Umeki
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan;
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Sako
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan; Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan; maruta@soka.ac.jp.
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Maruta
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D020837:SOS1 Protein / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; D020837:SOS1 Protein; D006160:Guanosine Triphosphate; C495971:Hras protein, mouse; D020559:Monomeric GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D009327:4-Chloro-7-nitrobenzofurazan
keywords: G-proteins; GEF; Ras; fluorescence; nucleotide analogue