Structural basis of the stability of equilibrium and kinetic intermediates in protein folding

The main aim of this project is to clearify the difference in stability , kinetics and the mechanism of folding of two homologous proteins, human stefins A and B. They belong to the cystatin superfamily of the cysteine proteinase inhibitors and have been prepared in recombinant form (in the JSI lab). They are physiologically important proteins, lately, their role in apoptosis and in certain diseases of the central neural system has been discovered. The two stefins differ in the mode of denaturation where stefin A behaves two-state and stefin B forms intermediates on acid and chemical denaturation (acid molten globule intermediate). Similarly, in the folding kinetics stefin A behaves two-state whereas stefin B populates intermediates. We intend to prepare certain hybrid and site-specific mutants and study influence of pH and perturbants (as for example TFE) on the stability and folding behavior. The other interesting point about stefins (and cystatins) is the fact that they form dimers under partially denaturing conditions. Along this project a more detailed study of stefin A dimerization (by CD , SEC and NMR - project of dr.R.Jerala) has been undertaken. Similar studies are planned for stefin B where in addition to the irreversible dimers, (a different kind of ?) dimers appear reversibly as a function of salt, protein concentration and pH. The property of some mutants not to dimerize (and aggregate) could potentially be useful for NMR studies and putative application of stefins ''''in vivo''''.