Kinetic Coupling of Folding and Prolyl Isomerization of β2-Microglobulin Studied by Mutational Analysis
Kinetic Coupling of Folding and Prolyl Isomerization of β2-Microglobulin Studied by Mutational Analysis
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DOI：
10.1016/j.jmb.2008.08.003
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发表时间：
2008-10-24
影响因子：
5.6
通讯作者：
Goto, Yuji
中科院分区：
文献类型：
--
作者：
Sakata, Michiko;Chatani, Eri;Goto, Yuji
β（2）-微球蛋白（β 2-m）是一种导致透析相关淀粉样变性的蛋白质，采用典型的免疫球蛋白结构域折叠，其N端肽键为顺式异构体中的Pro32。β 2-m的重折叠受到Pro32缓慢的反式-顺式异构化的限制，这意味着具有非天然反式-Pro32异构体的中间体是淀粉样蛋白原纤维形成的前体。为了进一步了解β 2-m的Pro限制性折叠，我们使用两种突变体（W39和P32 V β 2-ms）以及野生型β 2-m研究了Gdn-HCl依赖性去折叠/重折叠动力学。W39 β 2-m是一种三重突变体，其中两个真正的Trip残基（Trp 60和Trp 95）都被Phe取代，并且在Leu 39的位置引入了与其他免疫球蛋白结构域共有的埋藏的Trp（即，L39W/W60F/W95F）。W39 Q-m在折叠时表现出显著的荧光猝灭，使得能够详细分析Pro限制的解折叠/重折叠。另一方面，P32 V β 2-m是Pro 32被瓦尔取代的突变体，可用于探测Pro 32的反式至顺式异构化的动力学作用。这些突变体的展开/重折叠动力学的比较分析，包括三种类型的双跳实验显示脯氨酰异构化与构象转变相结合，导致明显不寻常的动力学，特别是对于展开。我们认为，仔细考虑去折叠/重折叠和脯氨酰异构化的动力学耦合，这在最近的研究中往往被忽视，是必不可少的澄清蛋白质折叠的机制，而且，它的生物学意义。(C)2008爱思唯尔有限公司版权所有。
beta(2)-Microglobulin (beta 2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of beta 2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of beta 2-m, we Studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V beta 2-ms) as well as the wild-type beta 2-m. W39 beta 2-m is a triple mutant in which both of the authentic Trip residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 Q-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V beta 2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these Mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic Coupling Of unfolding/refolding and prolyl isomerization which has tended to be neglected in recent Studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance. (C) 2008 Elsevier Ltd. All rights reserved.