DISSECTING THE STRUCTURE OF A PARTIALLY FOLDED PROTEIN - CIRCULAR-DICHROISM AND NUCLEAR-MAGNETIC-RESONANCE STUDIES OF PEPTIDES FROM UBIQUITIN
DISSECTING THE STRUCTURE OF A PARTIALLY FOLDED PROTEIN - CIRCULAR-DICHROISM AND NUCLEAR-MAGNETIC-RESONANCE STUDIES OF PEPTIDES FROM UBIQUITIN
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DOI：
10.1006/jmbi.1993.1600
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发表时间：
1993-11-20
影响因子：
5.6
通讯作者：
WOOLFSON, DN
中科院分区：
文献类型：
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作者：
COX, JPL;EVANS, PA;WOOLFSON, DN
泛素的部分有序形式（A 态）的结构元素的性质和相互作用已被研究，泛素在低 pH 值下在 40% 至 60% 的甲醇水溶液中填充。在相同条件下研究了两种合成肽：U(1-21)，对应于泛素天然 (N) 和 A 状态的 N 末端 β-发夹；U(1-35)，包括该发夹和 α-螺旋。圆二色性研究表明，虽然这些肽大部分在水中展开，但它们在 30% 和 60% 甲醇中的结构含量与相应的天然二级结构相当。 1H nmr 的序列特异性分配U(1-35) 在甲醇水溶液中的光谱和随后的二级结构测定证实了类似天然二级结构的细节保守性。发现U(1-35)、U(1-21)和A态本身的光谱中相应的共振具有非常相似的化学位移，这表明β-发夹独立存在于部分折叠的蛋白质中，很少或没有受到分子其余部分的影响。这通过结构元件之间的核欧沃豪塞增强效应的核欧沃豪塞增强光谱和旋转框架核欧沃豪塞增强光谱中的虚拟缺失证实了这一点。光盘。和核磁共振有证据表明，A 态分子 C 端一半的结构很大程度上是非天然的。因此，尽管甲醇对于在没有更广泛的天然相互作用的情况下确保其稳定性是必要的，但β-发夹的结构，包括其氢键的记录，似乎完全由其自身的序列决定。泛素序列第一部分的这种内在结构偏好比 C 端一半强得多，这一结论反映在各种二级结构预测算法的结果中。
The nature and interaction of structural elements in a partially ordered form of ubiquitin, the A-state, which is populated at low pH in 40 to 60% aqueous methanol, have been investigated. Two synthetic peptides have been studied under the same conditions: U(1-21), corresponding to the N-terminal β-hairpin in the native (N) and A-states of ubiquitin and U(1-35), which includes this hairpin plus an α-helix. Circular dichroism studies indicate that, although these peptides are largely unfolded in water, their structural content in 30 and 60% methanol is comparable with the corresponding native secondary structure. Sequence-specific assignments of the1H n.m.r. spectra of U(1-35) in aqueous methanol and subsequent secondary structure determination confirm the conservation in detail of native-like secondary structure. Corresponding resonances in spectra of U(1-35), U(1-21) and the A-state itself were found to have closely similar chemical shifts, suggesting that the β-hairpin exists independently in the partially folded protein, with little or no influence from the rest of the molecule. This is confirmed by the virtual absence in nuclear Overhauser enhancement spectroscopy and rotating frame nuclear Overhauser enhancement spectroscopy spectra of nuclear Overhauser enhancement effects between structural elements. c.d. and n.m.r. evidence suggests that structure in the C-terminal half of the molecule in the A-state is largely non-native. Thus, although methanol is necessary to assure its stability in the absence of wider native interactions, the structure of the β-hairpin, including the register of its hydrogen bonding, appears to be determined entirely by its own sequence. This intrinsic structural preference in the first part of the ubiquitin sequence is much stronger than in the C-terminal half, a conclusion reflected in the results from a variety of secondary structure prediction algorithms.