Staphylococcal nuclease (SNase) and its mutants were used as model proteins in this project. The task was to delineate folding mechanisms and provide energetic and dynamic information on folding intermediates of SNase and its mutants. Energetically SNase folding is a quasi-two-state process, i.e. there exist only U and N under all folding conditions (U being the unfolded state and N the native state). However, kinetics indicate that U is composed of U₁, U₂ and U₃ and N is composed of I_ia and N₀, the 3 Us being the sub-states of the unfolded protein and I and N the sub-states of the folded protein. I_ia, a highly structured, inactive protein, with no enzymatic activity. When cis-trans isomerization of proline residues (six prolines in SNase) is considered, the mechanism of SNase folding i...[ Read more ]

Staphylococcal nuclease (SNase) and its mutants were used as model proteins in this project. The task was to delineate folding mechanisms and provide energetic and dynamic information on folding intermediates of SNase and its mutants. Energetically SNase folding is a quasi-two-state process, i.e. there exist only U and N under all folding conditions (U being the unfolded state and N the native state). However, kinetics indicate that U is composed of U₁, U₂ and U₃ and N is composed of I_ia and N₀, the 3 Us being the sub-states of the unfolded protein and I and N the sub-states of the folded protein. I_ia, a highly structured, inactive protein, with no enzymatic activity. When cis-trans isomerization of proline residues (six prolines in SNase) is considered, the mechanism of SNase folding is represented by

U_3c↔U_2c↔U_1c↔ I_iac↔ N_0c

↕      ↕       ↕       ↕       ↕

U_3t ↔U_2t↔ U_1t↔ I_iat ↔ N_0t

The subscript c and t indicate that Pro117 exists, respectively, in the cis- and in the trans-isomeric forms. This project has determined free energy, enthalpy and entropy for each species and activation energy and rate constant for each kinetic step under strongly folding and strongly unfolding conditions. The present data have suggested that although free energy of intermediate states provides the driving force, pathway of folding is determined by the activation energy separating these intermediates. Folding is a descending toward the global free energy minimal of the native state via the least activation path (LAP) in the vast energy landscape. Cis/trans isomerization of six proline residues can shape the folding funnel although not every high barrier represents proline isomerization. Hydrophobic condensation, tertiary structure formation and other molecular events in protein folding are also examined and characterized.