Thermal denaturation of human cystatin C and two 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 measured 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 W106S cystatin C exhibits a higher melting temperature by 4 degrees than the wild-type protein. This contrasts with its reduced inhibitory activity and represents an example where activity changes are due to local effects and are not correlated to stability. From the ratio between Van't Hoff and calorimetric enthalpies it is judged that recombinant human cystatin C and Leu-9 cystatin C are dimeric prior to thermal unfolding whereas W106S cystatin C is monomeric. Melting temperatures and estimated stabilities for some other members of the cystatin superfamily 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 (Tm = 82 degrees C) is placed in between the more stable human stefin A (Tm = 95 degrees C) and the less stable human stefin B (Tm = 66 degrees C) whereas chicken cystatin behaves as a thermophilic protein, melting above 115 degrees C. To illustrate secondary structure changes, thermal denaturations of the recombinant human cystatin C and of W106S cystatin C were followed by circular dichroism in the far UV. It was found that the change in tertiary structure (revealed by DSC) precedes the major change in secondary structure.
Zerovnik
E
E
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Cimerman
N
N
Kos
J
J
Turk
V
V
Lohner
K
K
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002645:Chickens; D002942:Circular Dichroism; D055316:Cystatin C; D015891:Cystatins / Q000737:chemistry*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry
C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; C100158:cystatin, egg-white