Department of Biochemistry, Faculty of Pharmacy, University of Tuzla, Univerzitetska 1, 75000 Tuzla, Bosnia and Herzegovina.
Biochem Biophys Res Commun. 2011 Nov 18;415(2):337-41. doi: 10.1016/j.bbrc.2011.10.059. Epub 2011 Oct 18.
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.
研究了芳香族残基 75 位在蛋白质稳定性、折叠机制和淀粉样纤维形成机制中的作用，研究对象为人源 stefin B 变体（31 位为 Y）及其点突变 H75W。为了揭示折叠和聚集交叉点的构象，首先研究了人源 stefin B(Y31)变体的折叠和寡聚体形成的动力学。结果发现，在 pH4.8 和 10%TFE 下，该蛋白分三个动力学阶段折叠；pH 和溶剂条件在较长时间内将蛋白转化为淀粉样纤维。相同的 pH 值导致形成类似天然的中间态（先前对该变体的研究中已知），这意味着折叠和淀粉样纤维形成过程共享相同的结构中间态，在这种情况下为类似天然的二聚体。在 pH5.8 和 7.0 下，stefin B 在两个中间态的四个动力学阶段中折叠到天然状态。相比之下，突变体 H75W 无法完全折叠，在所有研究的 pH 值下都停留在中间态：4.8、5.8 和 7.0。在 pH4.8 和 5.8，突变体在一个动力学阶段折叠成“熔融球蛋白”类型的中间态，这得出结论，其折叠机制与相同 pH 值下的亲本 stefin B 不同。在 pH7.0 时，突变体 H75W 分三个动力学阶段折叠成类似天然的中间态，类似于 pH4.8 时 stefin B 的折叠。
