Zerovnik E, Virden R, Jerala R, Kroon-Zitko L, Turk V, Waltho J P
Department of Biochemistry & Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Proteins. 1999 Aug 1;36(2):205-16.
Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.
三氟乙醇（TFE）已被用于探究同源半胱氨酸蛋白抑制剂——人stefin A和B的天然态稳定性及折叠途径的差异。在4.5 mol/L盐酸胍中完全展开后，stefin A在pH 6.0、20℃及11%（体积/体积）TFE、0.75 mol/L盐酸胍的条件下重新折叠，在230和280 nm处获取圆二色（CD）信号的一级速率常数分别为4.1 s⁻¹和5.5 s⁻¹，几乎相同，这两个速率明显高于在无TFE时相同两个探针观察到的0.11 s⁻¹的值。通过酪氨酸荧光监测，在10%（体积/体积）TFE时重折叠速率加速最大。当在66%（体积/体积）TFE、pH 3.3条件下变性的stefin A重折叠至相同最终条件时，观察到相似的重折叠速率（在230和280 nm处椭圆率分别为6.2 s⁻¹和7.2 s⁻¹）。在3.45 mol/L盐酸胍中完全展开后，stefin B在pH 6.0、20℃及7%（体积/体积）TFE、0.57 mol/L盐酸胍的条件下重新折叠，在280 nm处椭圆率变化的速率常数为32.8 s⁻¹；该速率仅为无TFE时观察到的速率的两倍。与stefin A的一个主要区别在于，在TFE存在下stefin B的重折叠在230 nm处的椭圆率出现过冲，其值比天然蛋白中的值大10%；该信号缓慢弛豫（0.01 s⁻¹）至最终天然值，近紫外CD信号几乎没有伴随变化；这种变化的大部分在两个较快阶段。在42%（体积/体积）TFE、pH 3.3条件下变性后，重折叠至相同最终条件的动力学表现出相同的限速步骤（0.01 s⁻¹），但最初更快。结果表明，与stefin A类似，在TFE存在下stefin B更快地形成其疏水核心和三级结构的主要部分。结果表明stefin B的α-螺旋中间体具有高度结构。《蛋白质》1999年；36:205 - 216。
