通过氢交换和核磁共振光谱表征的葡萄球菌核酸酶折叠中间体。
Staphylococcal nuclease folding intermediate characterized by hydrogen exchange and NMR spectroscopy.
作者信息
Jacobs M D, Fox R O
机构信息
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
出版信息
Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):449-53. doi: 10.1073/pnas.91.2.449.
Abstract
Pulsed hydrogen-deuterium exchange during refolding was used to probe the protection of backbone amide hydrogens from solvent exchange of the staphylococcal nuclease Pro117-->Gly variant. The extent of exchange for 39 residues was determined by two-dimensional proton NMR after refolding for 5 ms to 10 s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed of two beta-sheets formed by local sequence interactions was observed after a 5-ms refolding period. Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors of 10-60) and almost no protection in the alpha-helices (protection factors of < 10). The pattern of labeling is consistent with a role for beta-turns and beta-hairpins in the formation of the early intermediate.
摘要
在重折叠过程中使用脉冲氢-氘交换来探测葡萄球菌核酸酶Pro117→Gly变体的主链酰胺氢免受溶剂交换的保护情况。在重折叠5毫秒至10秒后，通过二维质子核磁共振确定了39个残基的交换程度。推断出三个动力学阶段。在5毫秒的重折叠期后，观察到由局部序列相互作用形成的两个β-折叠组成的早期重折叠中间体中酰胺的适度保护。通过在重折叠100毫秒后改变高pH标记脉冲来确定保护因子。中间状态对β-折叠中的残基有适度但显著的保护（保护因子为10-60），而在α-螺旋中几乎没有保护（保护因子<10）。标记模式与β-转角和β-发夹在早期中间体形成中的作用一致。
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