Nucleotide binding to tubulin-investigations by nuclear magnetic resonance spectroscopy.
In an attempt to distinguish between the interaction of GTP and ATP with tubulin dimer, high-resolution 1H- and 31P-NMR experiments have been carried out on the nucleotides in the presence of tubulin. The location of the ATP binding sites on the protein in relation to the GTP sites is still not clear. Using NMR spectroscopy, we have tried to address this question. Evidence for the existence of a site labelled as X-site and another site (labelled as L-site) for both the nucleotides on tubulin has been obtained. It is suggested that this X-site is possibly the putative E-site. In order to gain further insight into the nature of these sites, the Mg(II) at the N-site has been replaced by Mn(II) and the paramagnetic effect of Mn(II) on the linewidth of the proton resonances of tubulin-bound ATP and GTP has been studied. The results show that the L-site nucleotide is closer to the N-site metal ion compared to the X-site nucleotide. On the basis of these results, it is suggested that the L-site of ATP is distinct from the L-site of GTP while the X-site of both the nucleotides seems to be same. By using the paramagnetic effect of the metal ion, Mn(II), at the N-site on the relaxation rates of tubulin-bound ATP at L-site, distances of the protons of the base, sugar and phosphorous nuclei of the phosphorous moiety of ATP, from the N-site metal ion have been mapped. The base protons are approximately equal to 0.8-1 nm from this metal ion site. On the other hand, the phosphorous nuclei of the phosphate groups are somewhat nearer (approximately equal to 0.4-0.5 nm) from the N-site metal ion.
Tata Institute of Fundamental Research, Colaba, Bombay, India.
S S
SS
Rai
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K
Kuchroo
S R
SR
Kasturi
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D006041:Goats; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000737:chemistry; D011485:Protein Binding; D013696:Temperature; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D014404:Tubulin; D008345:Manganese; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium