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 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.
Jerala
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Laboratory for Molecular Modeling and NMR Spectroscopy, National Institute of Chemistry, Ljubljana, Slovenia. roman.jerala@ki.si
Zerovnik
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D000577:Amides / Q000378:metabolism; D002942:Circular Dichroism; D055312:Cystatin A; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D000577:Amides; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011522:Protons; D011994:Recombinant Proteins; D006859:Hydrogen; C026407:CSTA protein, human