Folding thermodynamics and kinetics of the leucine-rich repeat domain of the virulence factor Internalin B

Although the folding of α-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing β-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single β-strand that forms a continuous β-sheet with neighboring repeats, and an N-terminal α-helical capping motif. Despite its modular structure, InlB folds in an equilibrium twostate manner, as reflected by the identical thermodynamic parameters obtained by monitoring its sigmoidal urea-induced unfolding transition by different spectroscopic probes. Although equilibrium two-state folding is common in α-helical repeat proteins, to date, InlB is the only β-sheet-containing repeat protein for which this behavior is observed. Surprisingly, unlike other repeat proteins exhibiting equilibrium two-state folding, InlB also folds by a simple two-state kinetic mechanism lacking intermediates, aside from the effects of prolyl isomerization on the denatured state. However, like other repeat proteins, InlB also folds significantly more slowly than expected from contact order. When plotted against urea, the rate constants for the fast refolding and single unfolding phases constitute a linear chevron that, when fitted with a kinetic two-state model, yields thermodynamic parameters matching those observed for equilibrium folding. Based on these kinetic parameters, the transition state is estimated to comprise 40% of the total surface area buried upon folding, indicating that a large fraction of the native contacts are formed in the rate-limiting step to folding. Published by Cold Spring Harbor Laboratory Press.