Evidence against a Permanently Folded Nucleation Sequence in the Intrinsically Disordered Protein Stathmin Measured by Site-Directed Spin Labeling EPR Spectroscopy

Abstract

After their relatively recent discovery and classification, it has been shown that intrinsically disordered proteins (IDPs) play essential roles in cellular regulation and signal transduction. Stathmin is a eukaryotic IDP responsible for regulating the polymerization equilibrium between the tubulin heterodimer and microtubules, and is also highly expressed in cancerous cells. Stathmin exists in a highly-ordered folded state when bound to its binding partner tubulin; however, alone in solution, as an IDP lacking those stabilizing binding interactions, stathmin is likely to explore conformational space in various equilibria between partially and completely folded states. Interestingly, it has been suggested that soluble stathmin has a short helical region (Glu55-Ala73) of persistent foldedness, and that this region might act as an α-helical ‘nucleation sequence' necessary for further folding in the C-terminal direction. Alternatively, we believe that the complete length of stathmin exists in a folding-unfolding equilibrium, and here we report data supporting this hypothesis. Nitroxide spin labeled mutants of stathmin throughout the Glu55-Ala73 region - rotationally immobilized through coupling to CNBr-activated sepharose beads - were studied by electron paramagnetic resonance (EPR) spectroscopy. The continuous wave EPR spectra for all 19 spin labeled mutants unambiguously indicate that this region of stathmin exists in a dynamic equilibrium between folded (ordered) and unfolded (disordered) states, and that no such helical nucleation sequence exists. We also show that this folding equilibrium in stathmin is shifted by the common biochemical viscosity modifiers sucrose, glycerol, and Ficoll-70.