Stathmin Exists as an Oligomer in Solution, as Evidenced by Static Light-Scattering, Native Gel Electrophoresis, and EPR Spectroscopy

Abstract

Intrinsically disordered proteins (IDPs) are an interesting class of highly dynamic, typically regulatory, proteins. They lack a native three-dimensional fold, but can often acquire a stable, ordered structure upon interaction with a binding partner. Various IDPs have been reported to exist in cells as ordered oligomers or disordered aggregates, often resulting in disease. In particular, stathmin is a regulatory IDP involved in the disassembly of cytoskeletal microtubules. As such, it is essential for proper cell function (i.e., processes coordinating the cell cycle, maintaining cell shape, etc.); improper regulation of stathmin activity has been linked to neurodegenerative diseases, mental disorders, and various cancers. It is thus important to study the solution-phase structure and conformational dynamics of stathmin, as they likely emulate the protein's behavior in cellular environments. Our preliminary static light scattering and native gel electrophoresis data suggest that stathmin may exist as an oligomer in solution, contradicting previous observations of a monomer by analytical ultracentrifugation. To investigate this further, we performed site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy on various singly-labeled stathmin mutants. The resulting EPR traces all exhibit a spectral component substantially broadened due to the dipolar interaction, implying the close proximity of two or more spin labels, likely due to dimerization or higher-order oligomerization. Upon dilution of the spin-labeled proteins with unlabeled wild-type stathmin, the spectral weight of the dipolar-broadened component was significantly reduced. The data collectively presented here support our hypothesis that stathmin exists as an oligomer in solution. These results have important implications on our understanding of the conformational dynamics of this IDP and the roles that oligomerized IDPs have in diseases.