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.
Auerswald
E A
EA
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgischen Klinik und Poliklinik, LMU München, Germany.
Nägler
D K
DK
Schulze
A J
AJ
Engh
R A
RA
Genenger
G
G
Machleidt
W
W
Fritz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015891:Cystatins / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D014644:Genetic Variation; D019791:Guanidine; D006146:Guanidines; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D017384:Sequence Deletion
D015891:Cystatins; D006146:Guanidines; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D019791:Guanidine