Abstract
Microtubules are biopolymers that perform diverse cellular functions. Microtubule behavior regulation occurs in part through post-translational modification of both the α- and β-subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and/or glutamate residues to the disordered C-terminal tails (CTTs) of tubulin. Because of their prevalence in stable, high-stress cellular structures such as cilia, we sought to determine if these modifications alter microtubules' intrinsic stiffness. Here, we describe the purification and characterization of differentially modified pools of tubulin from Tetrahymena thermophila. We found that post-translational modifications do affect microtubule stiffness but do not affect the number of protofilaments incorporated into microtubules. We measured the spin dynamics of nuclei in the CTT backbone by NMR spectroscopy to explore the mechanism of this change. Our results show that the α-tubulin CTT does not protrude out from the microtubule surface, as is commonly depicted in models, but instead interacts with the dimer's surface. This suggests that the interactions of the α-tubulin CTT with the tubulin body contributes to the stiffness of the assembled microtubule, thus providing insight into the mechanism by which polyglycylation and polyglutamylation can alter microtubule mechanical properties.