Φ-Analysis at the Experimental Limits: Mechanism of β-Hairpin Formation

Abstract

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded β-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0–7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 °C so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 μs–400 μs conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Φ-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Φ-values of 0 and others 1, as readily seen in chevron plots, with Φ-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 °C, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first β-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the β-strands in the denatured state and that these interactions provide the driving force for folding of the first β-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between β1 and β2 does a hydrogen bond form to stabilize the intervening turn, or the first β-turn.