PRODUCTION, INHIBITORY ACTIVITY, FOLDING AND CONFORMATIONAL-ANALYSIS OF AN N-TERMINAL AND AN INTERNAL DELETION VARIANT OF CHICKEN CYSTATIN

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chick en egg white cystatin gene in Escherichia coli. The variant des-Ser1-P ro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-de l 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-heli x II variant, shortened by 19 amino acids, is a basic, stefin-like min i-cystatin with one disulfide bridge. Its inhibitory properties are id entical to chicken cystatin and its stability against Gdn/HCl is simil ar. The folding of the del-helix II variant corresponds best to a sing le step process. In contrast to this, the reversible folding of natura l and recombinant chicken cystatin is more complex when recorded by ei ther tryptophan fluorescence or far-ultraviolet-CD. With increasing Gd n/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct i ntermediate states. The N-del 2 variant has a neutral pi and shows fol ding behaviour very similar to natural and recombinant chicken cystati n. However its inhibition constants with papain, actinidin and catheps in B and L are 1000-100000-fold higher than those obtained with natura l and recombinant chicken cystatin.