Cox J P, Evans P A, Packman L C, Williams D H, Woolfson D N
Cambridge Centre for Molecular Recognition, University Chemical Laboratory, U.K.
J Mol Biol. 1993 Nov 20;234(2):483-92. doi: 10.1006/jmbi.1993.1600.
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 beta-hairpin in the native (N) and A-states of ubiquitin and U(1-35), which includes this hairpin plus an alpha-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 the 1H 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 beta-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 beta-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.
泛素的一种部分有序形式即A态，在40%至60%的甲醇水溶液中于低pH值时存在，对其结构元件的性质及相互作用进行了研究。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）态和A态中的N端β-发夹结构；以及U(1 - 35)，它包含该发夹结构加上一个α-螺旋。圆二色性研究表明，尽管这些肽在水中基本未折叠，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。对U(1 - 35)在甲醇水溶液中的1H核磁共振谱进行序列特异性归属并随后确定二级结构，详细证实了类天然二级结构的保守性。发现U(1 - 35)、U(1 - 21)和A态本身的谱图中相应的共振峰具有非常相似的化学位移，这表明β-发夹结构在部分折叠的蛋白质中独立存在，受分子其余部分的影响很小或没有影响。这在核Overhauser增强光谱和旋转坐标系核Overhauser增强光谱中结构元件之间几乎不存在核Overhauser增强效应得到了证实。圆二色性和核磁共振证据表明，A态分子C端一半的结构在很大程度上是非天然的。因此，尽管甲醇对于在缺乏更广泛的天然相互作用时确保其稳定性是必要的，但β-发夹结构，包括其氢键的排列，似乎完全由其自身序列决定。泛素序列第一部分的这种内在结构偏好比C端一半要强得多，这一结论反映在各种二级结构预测算法的结果中。
