Rami B R, Udgaonkar J B
National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore 560065, India.
Biochemistry. 2001 Dec 18;40(50):15267-79. doi: 10.1021/bi011701r.
Equilibrium and kinetic characterization of the high pH-induced unfolding transition of the small protein barstar have been carried out in the pH range 7-12. A mutant form of barstar, containing a single tryptophan, Trp 53, completely buried in the core of the native protein, has been used. It is shown that the protein undergoes reversible unfolding above pH 10. The pH 12 form (the D form) appears to be as unfolded as the form unfolded by 6 M guanidine hydrochloride (GdnHCl) at pH 7 (the U form): both forms have similar fluorescence and far-UV circular dichroism (CD) signals and have similar sizes, as determined by dynamic light scattering and size-exclusion chromatography. No residual structure is detected in the D form: addition of GdnHCl does not alter its fluorescence and far-UV CD properties. The fluorescence signal of Trp 53 has been used to monitor folding and unfolding kinetics. The kinetics of folding of the D form in the pH range 7-11 are complex and are described by four exponential processes, as are the kinetics of unfolding of the native state (N state) in the pH range 10.5-12. Each kinetic phase of folding decreases in rate with increase in pH from 7 to 10.85, and each kinetic phase of unfolding decreases in rate with decrease in pH from 12 to 10.85. At pH 10.85, the folding and unfolding rates for any particular kinetic phase are identical and minimal. The two slowest phases of folding and unfolding have identical kinetics whether measured by Trp 53 fluorescence or by mean residue ellipticity at 222 nm. Direct determination of the increase in the N state with time of folding at pH 7 and of the D form with time of unfolding at pH 12, by means of double-jump assays, show that between 85 and 95% of protein molecules fold or unfold via fast pathways between the two forms. The remaining 5-15% of protein molecules appear to fold or unfold via slower pathways, on which at least two intermediates accumulate. The mechanism of folding from the high pH-denatured D form is remarkably similar to the mechanism of folding from the urea or GdnHCl-denatured U form.
在pH值7 - 12范围内，对小分子蛋白质巴司他汀（barstar）高pH诱导的去折叠转变进行了平衡和动力学表征。使用了一种巴司他汀突变体形式，其含有单个色氨酸Trp 53，该色氨酸完全埋藏在天然蛋白质的核心部位。结果表明，该蛋白质在pH值高于10时发生可逆去折叠。pH值为12的形式（D形式）似乎与在pH值7时由6 M盐酸胍（GdnHCl）展开的形式（U形式）一样完全展开：通过动态光散射和尺寸排阻色谱法测定，这两种形式具有相似的荧光和远紫外圆二色性（CD）信号，并且大小相似。在D形式中未检测到残留结构：添加GdnHCl不会改变其荧光和远紫外CD特性。Trp 53的荧光信号已用于监测折叠和去折叠动力学。在pH值7 - 11范围内D形式的折叠动力学很复杂，由四个指数过程描述，天然状态（N状态）在pH值10.5 - 12范围内的去折叠动力学也是如此。折叠的每个动力学阶段的速率随着pH值从7增加到10.85而降低，去折叠的每个动力学阶段的速率随着pH值从12降低到10.85而降低。在pH值10.85时，任何特定动力学阶段的折叠和去折叠速率相同且最小。无论是通过Trp 53荧光还是通过222 nm处的平均残基椭圆率测量，折叠和去折叠的两个最慢阶段都具有相同的动力学。通过双跳测定法直接测定在pH值7时折叠过程中N状态随时间的增加以及在pH值12时D形式随时间的去折叠情况，结果表明85%至95%的蛋白质分子通过两种形式之间的快速途径进行折叠或去折叠。其余5%至15%的蛋白质分子似乎通过较慢的途径进行折叠或去折叠，在这些途径上至少积累了两种中间体。从高pH变性的D形式折叠的机制与从尿素或GdnHCl变性的U形式折叠的机制非常相似。
