title: Solid-state 31P NMR investigation on the status of guanine nucleotides in paclitaxel-stabilized microtubules.
abstract: Microtubule dynamics is a target for many chemotherapeutic drugs. In order to understand the biochemical effects of paclitaxel on the GTPase activity of tubulin, the status of guanine nucleotides in microtubules was investigated by (31)P cross-polarization magic angle spinning (CPMAS) NMR. Microtubules were freshly prepared in vitro in the presence of paclitaxel and then lyophilized in sucrose buffer for solid-state NMR experiments. A (31)P CPMAS NMR spectrum with the SNR of 25 was successfully acquired from the lyophilized microtubule sample. The broadness of the (31)P spectral lines in the spectrum indicates that the molecular environments around the guanine nucleotides inside tubulin may not be as crystalline as reported by many diffraction studies. Deconvolution of the spectrum into four spectral components was carried out in comparison with the (31)P NMR spectra obtained from five control samples. The spectral analysis suggested that about 13% of the nucleotides were present as GTP and 37% as GDP in the β-tubulin (E-site) of the microtubules. It was found that most of the GDPs were present as GDP-Pi complex in the microtubules, which seems to be one of the effects of paclitaxel binding.
authors: Daegu Center, Korea Basic Science Institute, 80 Daehakro, Bukgu, Daegu, 702-701, Korea; Ochang Center, Korea Basic Science Institute, 162 Yeongudanjiro, Ochangeup, Cheongwongun, Chungcheongbukdo, 363-883, Korea.
authors: Ga Hyang
authors: GH
authors: Lee
authors: Su Young
authors: SY
authors: Oh
authors: Kwon Joo
authors: KJ
authors: Yeo
authors: Taslima
authors: T
authors: Ferdous
authors: Meeyeon
authors: M
authors: Cho
authors: Younkee
authors: Y
authors: Paik
mesh_terms: D020558:GTP Phosphohydrolases / Q000032:analysis / Q000737:chemistry*; D006150:Guanine Nucleotides / Q000032:analysis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008870:Microtubules / Q000737:chemistry*; D017239:Paclitaxel / Q000032:analysis / Q000737:chemistry*; D010759:Phosphorus Isotopes / Q000737:chemistry; D011485:Protein Binding; D050257:Tubulin Modulators / Q000737:chemistry*
chemical_list: D006150:Guanine Nucleotides; D010759:Phosphorus Isotopes; D050257:Tubulin Modulators; D020558:GTP Phosphohydrolases; D017239:Paclitaxel
keywords: 31P; CPMAS; GTP; NMR; microtubules; paclitaxel