Solid-state super(31)P NMR investigation on the status of guanine nucleotides in paclitaxel-stabilized microtubules

• Published in: Magnetic resonance in chemistry 2015-05, Vol.53 (5), p.330-336
• Main Authors: Lee, Ga Hyang, Oh, Su Young, Yeo, Kwon Joo, Ferdous, Taslima, Cho, Meeyeon, Paik, Younkee
• Format: Article
• Language: English
• Subjects: Binding; Biochemistry; Diffraction; Guanines; Nuclear magnetic resonance; Nucleotides; Spectra; Sucrose
• ISSN: 0749-1581; 1097-458X
• DOI: 10.1002/mrc.4183

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 super(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 super(31)P CPMAS NMR spectrum with the SNR of 25 was successfully acquired from the lyophilized microtubule sample. The broadness of the super(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 super(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 beta -tubulin (E-site) of the microtubules. It was found that most of the GDPs were present as GDP-P sub(i) complex in the microtubules, which seems to be one of the effects of paclitaxel binding. Copyright copyright 2015 John Wiley & Sons, Ltd. A 31P CPMAS NMR spectrum was successfully acquired from the lyophilized, paclitaxel-stabilized microtubules. GDPs were present as GDP-Pi complex in the microtubules, which seems to be one of the effects of paclitaxel binding.