Characterizing Induced-Conformational Changes in Intrinscially Disordered Proteins via Multi-Frequency EPR Spectroscopy

Abstract

Intrinsically disordered proteins (IDPs) contain little to no secondary or tertiary structure and are often essential in biological systems. Many IDPs undergo a conformational change, where structure is induced upon binding to its target protein. Due to their very nature, structural studies of IDPs are often challenging. Here, we show how a multi-frequency approach to site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy can be utilized to characterize the mobility and conformational changes of IDPs. We have applied this method to IA3, which is a 68 residue IDP whose unstructured-to-α-helical conformational transition has been extensively characterized by various biophysical techniques. We monitored the induced conformational change in the presence of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE), at both X-, and W-band frequencies. Analyses of the X-band EPR spectral line shapes reveal that the data report on global correlation time changes consistent with a two-state model of an unstructured system and the tumbling of a rigid helix; more detailed analyzes of the X-band spectral line shapes can provide site-specific information on the residue level. Analysis of the W-band EPR spectral line shapes, however, more directly reveal site-specific structural changes. Line shape simulations of the data at both frequencies should provide further information on the site-specific conformational changes occurring in the presence of TFE and are currently underway. Using IA3 as a model system, we show multi-frequency EPR can provide insight into structural changes occurring in IDP systems that are otherwise difficult to characterize.