Solution Structure of the Q41N Variant of Ubiquitin as a Model for the Alternatively Folded N2 State of Ubiquitin

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• 作者：Soichiro Kitazawa ; Tomoshi Kameda ; Maho Yagi-Utsumi ; Kenji Sugase ; Nicola J. Baxter ; Koichi Kato ; Michael P. Williamson ; Ryo Kitahara
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：March 19, 2013
• 年：2013
• 卷：52
• 期：11
• 页码：1874-1885
• 全文大小：727K
• 年卷期：v.52,no.11(March 19, 2013)
• ISSN：1520-4995

文摘

It is becoming increasingly clear that proteins transiently populate high-energy excited states as a necessary requirement for function. Here, we demonstrate that rational mutation based on the characteristics of the structure and dynamics of proteins obtained from pressure experiments is a new strategy for amplifying particular fluctuations in proteins. We have previously shown that ubiquitin populates a high-energy conformer, N₂, at high pressures. Here, we show that the Q41N mutation favors N₂: high-pressure nuclear magnetic resonance (NMR) shows that N₂ is 70% populated in Q41N but only 20% populated in the wild type at ambient pressure. This allows us to characterize the structure of N₂, in which 伪₁-helix, the following loop, 尾₃-strand, and 尾₅-strand change their orientations relative to the remaining regions. Conformational fluctuation on the microsecond time scale, characterized by ¹⁵N spin relaxation NMR analysis, is markedly increased for these regions of the mutant. The N₂ conformers produced by high pressure and by the Q41N mutation are quite similar in both structure and dynamics. The conformational change to produce N₂ is proposed to be a novel dynamic feature beyond the known recognition dynamics of the protein. Indeed, it is orthogonal to that seen when proteins containing a ubiquitin-interacting motif bind at the hydrophobic patch of ubiquitin but matches changes seen on binding to the E2 conjugating enzyme. More generally, structural and dynamic effects of hydrodynamic pressure are shown to be useful for characterizing functionally important intermediates.