The equilibrium folding pathway of staphylococcal nuclease: identification of the most stable chain-chain interactions by NMR and CD spectroscopy.
The equilibrium folding pathway of staphylococcal nuclease: identification of the most stable chain-chain interactions by NMR and CD spectroscopy.
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葡萄球菌核酸酶的平衡折叠途径：通过 NMR 和 CD 光谱鉴定最稳定的链-链相互作用。
DOI：
10.1021/bi00049a004
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Shortle,D
中科院分区：
文献类型：
--
作者：
Wang,Y;Shortle,D
1995年9月21日收到的修订版Mandarin pt摘要：在以前的报告中[Alexandrescu，A. T.，Abeygunawardana，C.，& Shortle，D.（1994）Biochemistry 33，1063-1072]，NMR方法用于表征A131 A中的残余结构，A131 A是葡萄球菌核酸酶的大片段，其在非变性条件下用作模型变性状态。的基础上，大量失踪的酰胺质子的残基，形成一个三股反平行/？在天然状态下的β-片，可以得出结论，这种β-弯曲可能在A131 A中高度填充，由于不同构象状态之间相对缓慢的交换而具有严重的谱线展宽。在本报告中，圆二色性光谱和NMR光谱的结果表明，在尿素浓度低于6 M时，链/32-β 3形成β发夹。来自与该β发夹相邻的几个疏水残基的酰胺质子共振与所有β 2-β 3残基一起消失，表明局部的非天然疏水相互作用可能有助于稳定β发夹。在低于3 M的浓度下，A131 A中来自链β 1的所有酰胺共振也消失，表明β 1可能与β 2-β 3发夹联合收割机结合形成天然样β弯曲。此外，疏水螺旋a. 2从0 M尿素中约30%的群体减少到6 M尿素中约10%-15%，而螺旋a1从0 M尿素中10%-15%的群体减少到6 M尿素中检测不到。第二个，明显不同的变性状态，WT核酸酶在pH 3.0和低盐的表征，揭示了这种低密度的酸变性状态是在低浓度的尿素A131 A结构相似。从这些和以前发表的数据，可以为葡萄球菌核酸酶构建一个稳定的平衡折叠途径，该途径描述了形成天然状态所涉及的链-链相互作用的相对强度和相互依赖性。在过去的几年中，人们已经清楚，当蛋白质的天然状态经历可逆变性时，并不是所有的链-链相互作用都丢失（jima，1989; Dill & Shortle，1991;多布森，1992; Shortle，1993）。在已经获得结构信息的少数情况下，这种持久的残留结构涉及沿着链沿着紧邻的残基之间的疏水相互作用（Neri等人，1992; Logan等人，1994; Alexandrescu等人，1994年a）。这种残余结构在蛋白质折叠的能量学中起着重要的作用。一方面，如果这些结构是天然样的，那么它们代表早期折叠元件，当多肽链向天然状态前进时，这些早期折叠元件可以在随后的步骤中保留。另一方面，如果这些残余结构不是天然的，它们必须在折叠可以继续之前以自由能的一些代价被撤销。换句话说，对最终折叠状态没有贡献的残余结构起到减缓折叠过程的作用，并通过降低自由基来间接地使天然状态不稳定。
Revised Manuscript Received September 21, 1995® abstract: In a previous report [Alexandrescu, A. T., Abeygunawardana, C., & Shortle, D.(1994) Biochemistry 33, 1063—1072], NMR methods were used to characterize the residual structure in A131A, a large fragment of staphylococcal nuclease that serves as a model denatured state under nondenaturing conditions. On thebasis of a large number of missing amide protons for the residues that form a threestrand antiparallel/? sheet in the native state, it was concluded that this (3 meander may be highly populated in A131A, with severe line broadening due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectroscopy and NMR spectroscopy indicate strands/32-(33 form a (3 hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this (3 hairpin disappear in concert with all of the (32-(33 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpin. At concentrations below 3 M, all amide resonances from strand (31 inA131A also disappear, suggesting that (31 may combine with the f32-(33 hairpin to form a native-like (3 meander. In addition, the hydrophobic helix a. 2 decreases from approximately 30% populationin 0 M urea to approximately 10%—15% at 6 M urea, whereas helix al goes from 10%—15% populatedin 0 M urea to undetectable in 6 M urea. Characterization of a second, distinctly different denatured state, WT nuclease at pH 3.0 and low salt, reveals that this low-density acid-denatured state is structurally similar to A131A at low concentrations of urea. From these and previously published data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and interdependencies of the chain—chain interactions involved in forming the native state.In the past few years, it has become clear that not all chain—chain interactions are lost when the native state of a protein undergo reversible denaturation (Kuwajima, 1989; Dill & Shortle, 1991; Dobson, 1992; Shortle, 1993). In the small number of cases in which structural information has been obtained, this persistent, residual structure involves hydrophobic interactions betweenresidues in close proximity along the chain (Neri et al., 1992; Logan et al., 1994; Alexandrescu et al., 1994a). Such residual structuremust play a significant role in the energetics of protein folding. On the one hand, if these structures are native-like, then they represent early folding elements which can be retained during subsequent steps as the polypeptide chain advances toward the native state. On the other hand, if these residual structures are not native-like, they must be undone at some cost in free energy before folding can continue. In other words, residual structure thatdoes not contribute tothe final folded state serves to slow the process of folding and destabilize the native state indirectly by lowering the free