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]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin.