THERMAL-DENATURATION OF HUMAN CYSTATIN-C AND 2 OF ITS VARIANTS - COMPARISON TO CHICKEN CYSTATIN

Thermal denaturation of the recombinant human cystatin C, an 8-residue shorter variant (Leu-9 cystatin C), and the W106S mutant were measure d using differential scanning calorimetry (DSC). The finding that Leu- 9 cystatin C is of similar stability to the full length protein is in accordance with its nearly normal inhibitory activity. The variant W10 6S cystatin C exhibits a higher melting temperature by 4 degrees than the wild-type protein. This contrasts with its reduced inhibitory acti vity and represents an example where activity changes are due to local effects and are not correlated to stability. From the ratio between V an't Hoff and calorimetric enthalpies it is judged that recombinant hu man cystatin C and Leu-9 cystatin C are dimeric prior to thermal unfol ding whereas W106S cystatin C is monomeric. Melting temperatures and e stimated stabilities for some other members of the cystatin superfamil y of the cysteine proteinase inhibitors are presented which have been recorded previously or were collected for this study (chicken cystatin ). It is concluded that thermal stability of human cystatin C (T-m = 8 2 degrees C) is placed in between the more stable human stefin A (T-m = 95 degrees C) and the less stable human stefin a (T-m = 66 degrees C ) whereas chicken cystatin behaves as a thermophilic protein, melting above 115 degrees C. To illustrate secondary structure changes, therma l denaturations of the recombinant human cystatin C and of W106S cysta tin C were followed by circular dichroism in the far UV. It was found that the change in tertiary structure (revealed by DSC) precedes the m ajor change in secondary structure.