The cross-road between the mechanisms of protein folding and aggregation; study of human stefin B and its H75W mutant.
The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the "molten globule" type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8.
Department of Biochemistry, Faculty of Pharmacy, University of Tuzla, Univerzitetska 1, 75000 Tuzla, Bosnia and Herzegovina.
Aida
A
Smajlović
Selma
S
Berbić
Eva
E
Žerovnik
D002942:Circular Dichroism; D055313:Cystatin B / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
C524994:CSTB protein, human; D006639:Histidine; D055313:Cystatin B; D014364:Tryptophan