HYDROGEN-EXCHANGE IN NATIVE AND ALCOHOL FORMS OF UBIQUITIN
HYDROGEN-EXCHANGE IN NATIVE AND ALCOHOL FORMS OF UBIQUITIN
复制标题
DOI：
10.1021/bi00161a019
复制
发表时间：
1992-11-24
期刊：
影响因子：
2.9
通讯作者：
BRIGGS, MS
中科院分区：
文献类型：
--
作者：
PAN, YQ;BRIGGS, MS
泛素在60%的甲醇溶液中在低pH值下采用非天然折叠结构。二维核磁共振（2D NMR）被用来测量泛素在天然和甲醇形式的骨架酰胺质子的氢交换速率，并表征泛素在甲醇状态下的结构。天然形式的泛素中质子的保护因子（实验测定的交换率与未折叠多肽计算的交换率之比）范围从小于10到大于10（5）。大多数被保护不被交换的质子位于氢键二级结构的区域。最强保护的骨架酰胺质子是构成疏水核的残基的那些。甲醇溶液中泛素的氢交换太快，无法通过2D NMR直接测量，因此采用了标记方案，其中当蛋白质在甲醇溶液中时发生与溶剂的交换。在所需的标记时间后，通过用水性缓冲液稀释来淬灭交换，并通过蛋白质的天然形式的H-1 NMR测量质子占据率。甲醇形式的泛素中质子的保护因子范围为2.6至42，所有受保护的质子均位于天然形式的氢键结构中。同样，最强保护的质子是疏水核中残基的质子。天然和甲醇形式的氢交换速率模式的比较表明，几乎所有的天然二级结构都以甲醇形式存在，但它几乎均匀地被4-6 kcal/mol破坏。一些关键的三级相互作用，指示协会的α螺旋和β折叠也是显而易见的。甲醇形式的遍在蛋白的结构与遍在蛋白重折叠途径上的中间体相似，但不相同（Briggs，M.美国，&罗德，H.等人（1992）Proc. Acad. Sci. U.S.A. 89，2017 -2021）。
Ubiquitin adopts a non-native folded structure in 60% methanol solution at low pH. Two-dimensional nuclear magnetic resonance (2D NMR) was used to measure the hydrogen-exchange rates of backbone amide protons of ubiquitin in both native and methanol forms, and to characterize the structure of ubiquitin in the methanol state. Protection factors (the ratios of experimentally determined exchange rates to the rates calculated for an unfolded polypeptide) for protons in the native form of ubiquitin range from less than 10 to greater than 10(5). Most of the protons that are protected from exchange are located in regions of hydrogen-bonded secondary structure. The most strongly protected backbone amide protons are those of residues comprising the hydrophobic core. Hydrogen exchange from ubiquitin in methanol solution was too rapid to measure directly by 2D NMR, so a labeling scheme was employed, in which exchange with solvent occurred while the protein was in methanol solution. Exchange was quenched by dilution with aqueous buffer after the desired labeling time, and proton occupancies were measured by H-1 NMR of the native form of the protein. Protection factors for protons in the methanol form of ubiquitin range from 2.6 to 42, with all protected protons located in hydrogen-bonded structure in the native form. Again, the most strongly protected protons are those of residues in the hydrophobic core. Comparison of the patterns of the hydrogen-exchange rates in the native and methanol forms indicates that almost all of the native secondary structure persists in the methanol form, but that it is almost uniformly destabilized by 4-6 kcal/mol. Some key tertiary interactions indicative of association of the alpha helix and beta sheet are also evident. The structure of ubiquitin in the methanol form is similar, but not identical, to intermediates on the refolding pathway of ubiquitin (Briggs, M. S., & Roder, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89,2017-2021).