Characterization of conformationally heterogeneous proteins by electron paramagnetic resonance spectroscopy

Abstract

The Anfinsen paradigm of representing a protein by a single conformer is challenged by the uncertainty predictions that come with AlphaFold models, which suggest a greater extent of disorder. Characterization of such conformation heterogeneity requires experimental approaches that do not depend on long-range order. Site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is such an approach. The double electron–electron resonance (DEER) technique can access site-pair distance distributions in the 15–100 Å range, directly informing on ensemble width. SDSL-EPR can be applied in cellular environments, and recent work indicates that protein disorder is even more pervasive than predicted by AlphaFold. This suggests that the Anfinsen paradigm should be replaced by an ensemble paradigm.