Kinetics of Folding and Binding of an Intrinsically Disordered Protein : The Inhibitor of Yeast Aspartic Proteinase YPrA

The 68 residue peptide IA3 is an intrinsically unstructured protein that serves as an endogenous inhibitor of the yeast aspartic proteinase A (YPrA). Although unstructured in free solution, IA3 forms an N-terminal α helix as it binds to YPrA, leading to subnanomolar inhibition of the protease. Equilibrium structural and inhibition studies provide little insight into the mechanism and kinetics of the coupled folding and binding interaction. We have used laser temperature jump spectroscopy to study the kinetics of folding of free IA3 and of the interaction between IA3 and YPrA. Inducing folding with trifluoroethanol cosolvent allows us to determine the folding rate (kf ≈ 0.3 (μs)−1) and the unfolding rate (ku ≈ 3 (μs)−1) for free IA3 in water at 25 °C. A substantially faster relaxation process is observed in the presence of the proteinase; this process appears to be the kinetic signature of an intermediate binding step in the coupled folding and binding interaction of IA3 and YPrA.