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Solid‐state 31 P NMR investigation on the status of guanine nucleotides in paclitaxel‐stabilized microtubules
Author(s) -
Lee Ga Hyang,
Oh Su Young,
Yeo Kwon Joo,
Ferdous Taslima,
Cho Meeyeon,
Paik Younkee
Publication year - 2015
Publication title -
magnetic resonance in chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.483
H-Index - 72
eISSN - 1097-458X
pISSN - 0749-1581
DOI - 10.1002/mrc.4183
Subject(s) - chemistry , microtubule , nucleotide , guanine , tubulin , gtp' , nmr spectra database , magic angle spinning , nuclear magnetic resonance spectroscopy , gtpase , solid state nuclear magnetic resonance , crystallography , paclitaxel , stereochemistry , spectral line , biochemistry , nuclear magnetic resonance , medicine , physics , surgery , chemotherapy , astronomy , biology , gene , enzyme , microbiology and biotechnology
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‐P i complex in the microtubules, which seems to be one of the effects of paclitaxel binding. Copyright © 2015 John Wiley & Sons, Ltd.