Denaturation of stefin B by GuHCl, pH and heat; evidence for molten globule intermediates.
GuHCl, pH and thermal denaturation of the recombinant stefin B was followed by circular dichroism (CD) and size-exclusion chromatography (SEC). CD at 277 nm was taken as an indicator of integral tertiary structure and CD at 222 nm as an indicator of the secondary structure. Compactness was expressed by the volumes of elution on a SEC (Superose 12) column. Data on equilibrium denaturation were recalculated to the fractions of the native state (fN). The results have shown that equilibrium intermediates of the molten globule type exist under conditions of low pH, high temperature or medium GuHCl concentrations, namely A, T and G. Recent findings on structure and energetics of molten globule intermediates are reviewed.
Zerovnik
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Department of Biochemistry, J. Stefan Institute, University of Ljubljana, Slovenia.
Jerala
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Kroon-Zitko
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Turk
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Pain
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D002942:Circular Dichroism; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D015891:Cystatins; D006146:Guanidines; D011994:Recombinant Proteins; D055313:Cystatin B; D019791:Guanidine