ABSTRACT
p25alpha is a 219-residue protein which stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25alpha has been proposed to be natively unfolded, so that folding is coupled to interaction with its physiological partners. Here we show that recombinant human p25alpha is folded under physiological conditions, since it has a well structured and solvent-sequestered aromatic environment and considerable chemical shift dispersion of amide and aliphatic protons. With increasing urea concentrations, p25alpha undergoes clear spectral changes suggesting significant loss of structure. p25alpha unfolds cooperatively in urea according to a simple two-state transition with a stability in water of approximately 5 kcal/mol. The protein behaves as a monomer and refolds with a transient on-pathway folding intermediate. However, high sensitivity to proteolytic attack and abnormal gel filtration migration behavior suggests a relatively extended structure, possibly organized in distinct domains. A deletion mutant of p25alpha lacking residues 3-43 also unfolds cooperatively and with similar stability, suggesting that the N-terminal region is largely unstructured. Both proteins undergo significant loss of structure when bound to monomeric tubulin. The stoichiometry of binding is estimated to be 3-4 molecules of tubulin per p25alpha and is not significantly affected by the deletion of residues 3-43. In conclusion, we dismiss the proposal that p25alpha is natively unfolded, although the protein is relatively flexible. This flexibility may be linked to its tubulin-binding properties.
