The interaction of [¹³C]-enriched colchicine with tubulin as determined by NMR spectroscopy

[¹³C]Colchicine, labeled at the tropolone ring methoxy carbon, was used to study interactions with tubulin containing either Mg²⁺ or Mn²⁺ at the high affinity divalent cation binding site. Similar experiments were carried out in the presence of excess free divalent cation. The results show that: (1) when Mn²⁺ occupies the N-site, the ¹³C signal of the colchicine methoxy carbon of protein-bound colchicine is not broadened, indicating that in protein-bound colchicine the tropolone methoxy group is not close to the N-site cation; (2) when excess Mn²⁺ is present in solution this ¹³C signal is severely broadened, indicating that a low affinity divalent cation site or the exchangeable site (E-site) divalent cation is situated near the colchicine binding site; and (3) in the absence of paramagnetic ions a downfield chemical shift is observed for the tropolone methoxy carbon of colchicine upon binding to tubulin, suggesting that colchicine binds near an aromatic group(s) on tubulin.