Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions

Stefin A folds as a monomer under strongly native conditions. We have obser ved that under partially denaturing conditions in the temperature range fro m 74 to 93 degrees C it folds into a dimer, while it is monomeric above the melting temperature of 95 degrees C. Below 74 degrees C the dimer is trapp ed and it does not dissociate. The dimer is a folded and structured protein as judged by CD and NMR, nevertheless it is no more functional as an inhib itor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparabl e to the unfolding enthalpy. A large and negative dimerization enthalpy of -111(+/-8) kcal/mol was calculated from the temperature dependence of Be di ssociation constant. An irreversible pretransition at 10-15 deg. below the global unfolding temperature has been observed previously by DSC and can no w be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using N-15 isot opically enriched protein. The dimer is symmetric and the chemical shift di fferences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydr ogen exchange protection factors of the residues affected by dimer formatio n are higher in the dimer than in the monomer. The monomer to dimer transit ion is accompanied by a rapid exchange of all of the amide protons which ar e protected in the dimer, indicating that the transition state is unfolded to a large extent. Our results demonstrate that the native monomeric state of stefin A is actually metastable but is favored by the kinetics of foldin g. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions. (C) 1999 Academic P ress.