RESUMO
The intrinsically disordered protein (IDP) stathmin plays an important regulatory role in cytoskeletal maintenance through its helical binding to tubulin and microtubules. However, it lacks a stable fold in the absence of its binding partner. Although stathmin has been a focus of research over the past two decades, the solution-phase conformational dynamics of this IDP are poorly understood. It has been reported that stathmin is purely monomeric in solution and that it bears a short helical region of persistent foldedness, which may act to nucleate helical folding in the C-terminal direction. Here we report a comprehensive study of the structural equilibria local to this region in stathmin that contradicts these two claims. Using the technique of electron paramagnetic resonance (EPR) spectroscopy on spin-labeled stathmin mutants in the solution-phase and when immobilized on Sepharose solid support, we show that all sites in the helical nucleation region of stathmin exhibit multiple spectral components that correspond to dynamic states of differing mobilities and stabilities. Importantly, a state with relatively low mobility dominates each spectrum with an average population greater than 50%, which we suggest corresponds to an oligomerized state of the protein. This is in contrast to a less populated, more mobile state, which likely represents a helically folded monomeric state of stathmin, and a highly mobile state, which we propose is the random coil conformer of the protein. Our interpretation of the EPR data is confirmed by further characterization of the protein using the techniques of native and SDS PAGE, gel filtration chromatography, and multiangle and dynamic light scattering, all of which show the presence of oligomeric stathmin in solution. Collectively, these data suggest that stathmin exists in a diverse equilibrium of states throughout the purported helical nucleation region and that this IDP exhibits a propensity toward oligomerization.