Kotik M, Radford S E, Dobson C M
Oxford Centre for Molecular Sciences, University of Oxford, U.K.
Biochemistry. 1995 Feb 7;34(5):1714-24. doi: 10.1021/bi00005a028.
The folding of hen egg white lysozyme is complex, involving parallel pathways and distinct folding domains [Radford, S.E., Dobson, C.M., & Evans, P.A. (1992) Nature 358, 302-307]. In the present work the refolding of this protein from two denatured states that have different conformational properties, one generated by the presence of guanidinium chloride (GdmCl) and the other by dimethyl sulfoxide (DMSO), has been examined. Refolding was initiated by rapid dilution and followed by hydrogen-exchange pulse labeling, stopped-flow circular dichroism (CD) in the near-ultraviolet region, and stopped-flow fluorescence experiments. When the final refolding conditions were identical (545 mM GdmCl, 8% (v/v) DMSO, and 20 mM sodium acetate, pH 5.5, 20 degrees C), the folding behavior from the different denatured states monitored by near-UV CD and hydrogen-exchange pulse labeling was indistinguishable. These experiments indicate that the folding process of hen lysozyme is not significantly dependent on the nature of the two denatured states. The complexities in the pathway, therefore, appear to arise from properties of the collapsed state which is formed within the first few milliseconds of refolding. The kinetics of folding were found to be dependent on the concentration of DMSO in the final refolding buffer, although the fundamental properties of the pathway, including the existence of parallel events and distinct folding domains, are preserved under all the conditions studied. Inclusion of DMSO in the refolding buffer increases the rate of formation of native-like structure and of the native state itself. This could result from destablization of species formed early in folding, allowing them to rearrange more rapidly to permit productive folding to proceed. The results indicate that examination of a wide range of conditions will contribute substantially to a more complete understanding of protein folding pathways.
鸡蛋清溶菌酶的折叠过程很复杂，涉及平行途径和不同的折叠结构域[拉德福德，S.E.，多布森，C.M.，& 埃文斯，P.A.（1992年）《自然》358, 302 - 307]。在本研究中，对该蛋白质从两种具有不同构象性质的变性状态进行了重折叠研究，一种是由氯化胍（GdmCl）诱导产生的，另一种是由二甲基亚砜（DMSO）诱导产生的。重折叠通过快速稀释启动，随后进行氢交换脉冲标记、近紫外区域的停流圆二色性（CD）以及停流荧光实验。当最终重折叠条件相同时（545 mM GdmCl、8%（v/v）DMSO、20 mM 醋酸钠，pH 5.5，20℃），通过近紫外 CD 和氢交换脉冲标记监测到的来自不同变性状态的折叠行为没有区别。这些实验表明鸡蛋清溶菌酶的折叠过程并不显著依赖于这两种变性状态的性质。因此，途径中的复杂性似乎源于在重折叠最初几毫秒内形成的塌缩状态的性质。虽然在所研究的所有条件下，途径的基本性质（包括平行事件和不同折叠结构域的存在）都得以保留，但发现折叠动力学依赖于最终重折叠缓冲液中 DMSO 的浓度。在重折叠缓冲液中加入 DMSO 会增加类天然结构和天然状态本身的形成速率。这可能是由于折叠早期形成的物种不稳定，使其能够更快速地重新排列以促进有效折叠的进行。结果表明，对广泛条件的研究将极大地有助于更全面地理解蛋白质折叠途径。
