Folding studies of the cysteine proteinase inhibitor--human stefin A.
Reversible GuHCl denaturation of human stefin A (25 degrees C, pH 8) was monitored by the tyrosine fluorescence, by circular dichroism in the near UV and by circular dichroism in the far UV. In each case a midpoint of 2.8 +/- 0.1 M GuHCl was obtained, demonstrating the cooperativity of the denaturation. Kinetics of the slow folding on diluting the protein from the GuHCl denatured state, was also measured by the three spectroscopic probes (10 degrees C, pH 8). Results conform to a sequential mechanism. Denaturant concentration and temperature dependence of the slow folding were measured by fluorescence. From a linear Arrhenius plot the Ea of 100 +/- 5 kJ/mol was read. 'Double mixing' experiments revealed a slow reaction going on in the unfolded state which influenced the amplitude of the fluorescence changes. 'Double mixing' experiments performed by FPLC have shown that the folding itself, i.e., the formation of a compact state, was not dependent on the time spent under unfolding conditions.
Zerovnik
E
E
Department of Biochemistry, J. Stefan Institute, University of Ljubljana, Yugoslavia.
Lenarcic
B
B
Jerala
R
R
Turk
V
V
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D055312:Cystatin A; D015891:Cystatins / Q000737:chemistry* / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011257:Pregnancy Proteins / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D014466:Ultraviolet Rays
D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D006146:Guanidines; D011257:Pregnancy Proteins; C026407:CSTA protein, human; D011392:Proline; D019791:Guanidine