On the Origins of the Weak Folding Cooperativity of a Designed ββα Ultrafast Protein FSD-1

FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96) coupled with the implicit solvent model (IGB = 5), we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K) of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K) as well as the denatured structures. Author Summary: The protein folding process, in which a linear chain of amino acids reaches its biologically active three-dimensional shape, is fundamental to life. Small “ultrafast” folders, proteins that fold in microseconds, have received considerable attention, because these proteins serve as model systems for the folding of larger proteins, and thus permit a testing of the accuracy of computational models as well as an assessment of protein folding theories. FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for mixed α/β fold proteins. The suitability of this protein to describe the folding of naturally occurring proteins has however recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Prior simulations have not been successful in providing an interpretation of this broad melting transition. In the present study, our extensive molecular dynamics simulations using the AMBER protein force field (ff96) coupled with the implicit solvent model (IGB = 5) shed new light on the nature of the folding transition of this protein, as well as reveal the strengths and weaknesses of the force field in predicting the thermodynamics and kinetics of folding.