Direct Measurement of Nucleation and Growth Rates in Lysozyme Folding

Abstract

A kinetic folding intermediate of hen lysozyme is shown to form in a nucleation/growth type of mechanism. Under native solvent conditions, a nucleated state is formed slowly during refolding (τ = 14 ± 1 ms at 0 M GdmCl) and is rapidly converted to the folding intermediate (τ = 300 ± 150 μs at 0 M GdmCl). Under these conditions the nucleated state represents a high-energy state compared to the folding intermediate (ΔΔG⁰ = 13.7 ± 3 kJ/mol). At elevated concentrations of GdmCl, the nucleated state becomes more stable than the intermediate and it consequently becomes transiently populated during unfolding of the intermediate state. This allowed us to measure the rate constant of the growth step using stopped-flow double-jump experiments. At high concentrations of GdmCl (>5 M), the growth step becomes rate-limiting in unfolding, leading to the frequently observed rollover in the GdmCl dependence of the logarithm of the apparent rate constant of the unfolding reaction.