Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase
Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase
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DOI：
10.1016/j.jmb.2010.11.052
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发表时间：
2011-02-04
影响因子：
5.6
通讯作者：
Takahashi, Satoshi
中科院分区：
文献类型：
--
作者：
Konuma, Tsuyoshi;Kimura, Tetsunari;Takahashi, Satoshi
使用时间分辨小角 X 射线散射 (SAXS) 研究了芽孢杆菌RNA酶在折叠过程中的结构变化。芽孢杆菌RNA酶的折叠涉及突发阶段中间体（有时称为生理条件下的变性状态）、D-phys和第二个隐藏中间体。平衡 SAXS 测量表明，胍未折叠状态 (U) 的回转半径 (R-g) 为 26.9 +/- 0.7 埃，在较宽的变性剂浓度范围内基本保持恒定。时间分辨 SAXS 测量表明，从动力学 R-g 数据推断到零时间 R-g,R-0 的 Rg 值为 24.3 +/- 0.1 埃，该值小于 U 的值，但从具有相似链长的其他蛋白质的折叠中间体的值（19 埃）扩展而来。在爆发相变化之后，观察到 R-g(2) 的单指数减少，这对应于包含天然反式脯氨酸异构体的主要成分的天然状态的形成。我们估计含有非天然顺式脯氨酸异构体 (D-phys,D-cis) 的 D-phys 次要成分的 R-g 为 25.7 +/- 0.6 埃。此外，基于R-g、R-0，含有天然脯氨酸异构体(D-phys、D-tra)的Dphys主要成分的R-g估计为23.9+/-0.2埃。因此，芽孢杆菌RNA酶爆发相中间体的两个成分（D-phys，D-tra和D-phys，D-cis）仍然很大程度上扩展。据推测，D-phys 具有 N 末端螺旋和独立形成的 β 折叠中心，并且蛋白质其余部分的形成发生在较慢的阶段。 (C) 2010 Elsevier Ltd. 所有紧身衣保留。
Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D-phys and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R-g) of the guanidine unfolded state (U) is 26.9 +/- 0.7 angstrom, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the Rg value extrapolated from kinetic R-g data to time zero, R-g,R-0, is 24.3 +/- 0.1 angstrom, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 angstrom). After the burst-phase change, a single-exponential reduction in R-g(2) was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R-g of the minor component of D-phys, containing the non-native cis proline isomer (D-phys,D-cis) to be 25.7 +/- 0.6 angstrom. Moreover, R-g of the major component of Dphys containing the native proline isomer (D-phys,D-tra) was estimated as 23.9 +/- 0.2 angstrom based on R-g,R-0. Consequently, both components of the burst-phase intermediate of barnase (D-phys,D-tra and D-phys,D-cis) are still largely expanded. It was inferred that D-phys possesses the N-terminal helix and the center of the beta-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase. (C) 2010 Elsevier Ltd. All tights reserved.