Equilibrium and Kinetic Folding of Pigeon Lysozyme

In the present study, the search for a possible intermediate state in pigeon lysozyme is addressed by equilibrium and kinetic experiments using static and stopped-flow fluorescence and circular dichroism spectroscopies. In equilibrium conditions at pH 7.5, pigeon lysozyme shows no populated intermediate state in temperature- and GdnHCl-induced unfolding experiments. In the unfolding process at low pH, however, a distinct intermediate state with molten globule characteristics is observed. Ca2+ binding to the protein is found to stabilize the native state. The early folding intermediate observed in kinetic experiments corresponds to the equilibrium intermediate in that an important amount of secondary structure has already been established. Full accomplishment of native tertiary contacts is achieved in a fast exponential process with a rate constant (0.23−135 s-1) that is strongly dependent on refolding conditions. Binding experiments with the fluorescent inhibitor MeU-diNAG support these conclusions. The f...