Simulations and Experiments Provide a Convergent View of Protein Unfolded States under Folding Conditions

An accurate description of unfolded states under folding conditions is important for quantitative studies of protein stability, protein folding kinetics, and an understanding of how features of the unfolded state may have contributed to the convergent evolution toward foldable sequences. Here, we present results from multi-pronged biophysical investigations that uncover a comprehensive description of conformational ensembles accessed by unfolded states of proteins under folding conditions. We combined results from experiments based on time-resolved Forster resonance energy transfer, using multiple non-perturbing, amino-acid-sized dye pairs, and time-resolved small angle X-ray scattering, with novel molecular simulations and polymer physics theories. Previous work showed that aqueous milieus are poor solvents for polypeptide backbones. This should drive chain collapse and globule formation. Our current results, obtained for the N-terminal domain of the ribosomal protein L9, show that under folding conditions, native and non-native intra-chain interactions lead to transient globule formation, while large conformational fluctuations brought about by favorable sidechain-solvent interactions lead to an unfolded ensemble that behaves statistically like a flexible polymer would in a theta-solvent. Therefore, the unfolded state under folding conditions may be described as an ensemble with fluctuating elements of native and non-native non-random elements of structure that are averaged over to yield random coil-like statistics of chains in theta solvents. This behavior derives from the sequence-encoded interplay between backbone- and sidechain-mediated intra-chain and chain-solvent interactions. Our results have broad implications for interactions of unfolded proteins in crowded milieus, for folding-unfolding transitions, and for tilting the balance toward heterogeneous ensembles as is the case with intrinsically disordered proteins.