Probing the Conformational Dynamics of the Disordered 4E-BP2 Protein in Different Phosphorylation States using Single-Molecule Fluorescence

Abstract

Intrinsically disordered proteins (IDPs) are a class of proteins that lack a well-defined 3D structure while carrying out a diverse range of biological functions. They play a crucial role in regulatory pathways and in mediating interactions with multiple partners. Cap-dependent initiation of translation is regulated by the interaction of 120-residue eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Fluorescence correlation spectroscopy (FCS) and fluorescence anisotropy decay (FAD) were used to study the conformations, dynamics and binding of 4E-BP2. Anisotropy data informed the local chain flexibility at various points within the 4E-BP2. The segmental flexibility is hindered in the folded 18-62 region upon phosphorylation, whereas the rest of the chain becomes more flexible. The local segments become more flexible upon denaturation, which suggests that the native protein, whereas not having a stable 3D fold, contains significant secondary structure. Segmental rotational correlation times and wobbling cone angles extracted for different labelling sites along the chain provide a rigidity map of 4E-BP2 and are essential for elucidating the binding mode to eIF4E. Longer range intra-chain reorganization dynamics were assessed by FCS via photoblinking/quenching of the fluorophore by aromatic residues. Heterogeneous quenching kinetics were observed: multi-site phosphorylation of the protein slows the proximal chain motions and modulates the kinetics of the distal regions. Our results paint a complex behavior of 4E-BP2 upon phosphorylation and binding and suggest that electrostatics play a crucial role in modulating its dimensions and compactness and thus its activity.