Forge V, Wijesinha R T, Balbach J, Brew K, Robinson C V, Redfield C, Dobson C M
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QT, UK.
J Mol Biol. 1999 May 14;288(4):673-88. doi: 10.1006/jmbi.1999.2687.
The refolding of bovine alpha-lactalbumin (BLA) from its chemically denatured state in 6 M GuHCl has been investigated by a variety of complementary biophysical approaches. CD experiments indicate that the species formed in the early stages of refolding of the apo-protein have at least 85 % of the alpha-helical content of the native state, and kinetic NMR experiments show that they possess near-native compactness. Hydrogen exchange measurements using mass spectrometry and NMR indicate that persistent structure in these transient species is located predominantly in the alpha-domain of the native protein and is similar to that present in the partially folded A-state formed by the protein at low pH. The extent of the exchange protection is, however, small, and there is no evidence for the existence of well-defined discrete kinetic intermediates of the type populated in the refolding of the structurally homologous c-type lysozymes. Rather, both mass spectrometric and NMR data indicate that the rate-determining transition from the compact partially structured (molten globule) species to the native state is highly cooperative. The data show that folding in the presence of Ca2+ is similar to that in its absence, although the rate is increased by more than two orders of magnitude. Sequential mixing experiments monitored by fluorescence spectroscopy indicate that this slower folding is not the result of the accumulation of kinetically trapped species. Rather, the data are consistent with a model in which binding of Ca2+ stabilizes native-like contacts in the partially folded species and reduces the barriers for the conversion of the protein to its native state. Taken together the results indicate that folding of BLA, in the presence of its four disulphide bonds, corresponds to one of the limiting cases of protein folding in which rapid collapse to a globule with a native-like fold is followed by a search for native-like side-chain contacts that enable efficient conversion to the close packed native structure.
已通过多种互补的生物物理方法研究了牛α-乳白蛋白（BLA）在6 M盐酸胍中从化学变性状态的重折叠过程。圆二色性实验表明，脱辅基蛋白重折叠早期形成的物种具有至少85%的天然状态α-螺旋含量，动力学核磁共振实验表明它们具有接近天然的紧密性。使用质谱和核磁共振进行的氢交换测量表明，这些瞬态物种中的持久结构主要位于天然蛋白质的α结构域，并且与蛋白质在低pH下形成的部分折叠A状态中存在的结构相似。然而，交换保护的程度较小，没有证据表明存在结构同源的c型溶菌酶重折叠过程中出现的那种明确的离散动力学中间体。相反，质谱和核磁共振数据均表明，从紧密的部分结构化（熔球）物种到天然状态的速率决定转变是高度协同的。数据表明，在存在Ca2+的情况下折叠与不存在Ca2+时相似，尽管速率提高了两个以上数量级。通过荧光光谱监测的顺序混合实验表明，这种较慢的折叠不是动力学捕获物种积累的结果。相反，数据与一个模型一致，即Ca2+的结合稳定了部分折叠物种中的类似天然的接触，并降低了蛋白质转化为天然状态的障碍。综合结果表明，在其四个二硫键存在的情况下，BLA的折叠对应于蛋白质折叠的一种极限情况，即快速折叠成具有类似天然折叠的球体，随后寻找能够有效转化为紧密堆积天然结构的类似天然的侧链接触。
