Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: Energetics of peptide association and folding
Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: Energetics of peptide association and folding
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DOI：
10.1021/bi000718r
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发表时间：
2000-10-10
期刊：
影响因子：
2.9
通讯作者：
Searle, MS
中科院分区：
文献类型：
--
作者：
Jourdan, M;Searle, MS
对应于牛泛素N-和C-末端部分的肽片段U（1-35）和U（36-76）通过NMR显示在溶液中缔合形成适度稳定性的复合物（K-assn在pH 7.0时约为1.4 x 10（5）M-1），具有天然样结构的NMR特征。该复合物经历冷变性，与温度依赖性的估计，从NMR表明一个Δ C（p）度的片段络合的稳定性与确定的天然泛素的良好协议，这表明，在埋葬一个类似的疏水性表面积的片段协会的结果。复合物的稳定性显示出明显的pH依赖性，表明蛋白质表面上的离子相互作用有显着贡献。然而，在盐酸胍（Gdn . HCl）显示出很小的pH依赖性，这表明离子相互作用可以被变性剂“屏蔽”，如最近所建议的。对分离的肽片段的构象的检查显示了N-末端β-发夹（残基1-17）中天然样结构的低群体和螺旋片段（残基21-35）中弱新生螺旋倾向的证据。相比之下，C-末端肽（36-76）在水溶液中显示出非天然phi和psi角的证据，来自一些热化学位移;非天然α-螺旋结构在有机共溶剂存在下容易诱导，表明天然泛素和折叠片段复合物中的三级相互作用强烈决定了其结构偏好。数据表明，N-末端片段（1-35），其中螺旋和发夹之间的相互作用需要最小的构象熵损失，可以提供片段复合的成核位点。
Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (K-assn approximate to 1.4 x 10(5) M-1 at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a Delta C(p)degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn . HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Hot chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.