Author/Editor		Jerala, Roman; Žerovnik, Eva
Title		Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions
Type		članek
Source		J Mol Biol
Vol. and No.		Letnik 291, št. 5
Publication year		1999
Volume		str. 1079-89
Language		eng
Abstract		Stefin A folds as a monomer under strongly native conditions. We have observed that under partially denaturing conditions in the temperature range from 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 trapped 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 inhibitor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparable to the unfolding enthalpy. A large and negative dimerization enthalpy of -111(+/- 8) kcal/mol was calculated from the temperature dependence of the dissociation constant. An irreversible pretransition at 10-15 deg. below the global unfolding temperature has been observed previously by DSC and can now be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using 15N isotopically enriched protein. The dimer is symmetric and the chemical shift differences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydrogen exchange protection factors of the residues affected by dimer formation are higher in the dimer than in the monomer. The monomer to dimer transition is accompanied by a rapid exchange of all of the amide protons which are 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 folding. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions.
Descriptors		CYSTATINS CYSTEINE PROTEINASE INHIBITORS HYDROGEN DIMERIZATION AMIDES CIRCULAR DICHROISM THERMODYNAMICS TEMPERATURE RECOMBINANT PROTEINS PROTONS PROTEIN STRUCTURE, SECONDARY PROTEIN FOLDING PROTEIN DENATURATION NUCLEAR MAGNETIC RESONANCE MODELS, MOLECULAR KINETICS