Experimental test of the thermodynamic model of protein cooperativity using temperature-induced unfolding of a Ubq-UIM fusion protein.
Experimental test of the thermodynamic model of protein cooperativity using temperature-induced unfolding of a Ubq-UIM fusion protein.
复制标题
使用 Ubq-UIM 融合蛋白的温度诱导解折叠来实验测试蛋白质协同性的热力学模型。
DOI：
10.1021/bi101163u
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Makhatadze,GeorgeI
中科院分区：
文献类型：
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作者：
Patel,MayankM;Sgourakis,NikolaosG;Garcia,AngelE;Makhatadze,GeorgeI
本研究描述了一个Ubq-UIM融合构建体（Ubq-UIM）的热力学特性，设计从泛素-UIM相互作用系统，以确定它是否表现出折叠的协同性。Ubq-UIM融合构建体表现出比核心Ubq分子更高的稳定性，这与UIM螺旋与Ubq对接的发现一致。通过DSC和远紫外和近紫外CD光谱监测Ubq−UIM的温度诱导解折叠曲线。Ubq-UIM似乎表现出合作展开的两态模型的全球拟合的结果表明，远，近紫外CD和DSC热展开数据。通过选择性稳定或不稳定Ubq、UIM和/或界面的氨基酸取代进一步测试Ubq−UIM解折叠的协同性。这些取代对Ubq−UIM热力学性质的影响可以通过蛋白质协同性的热力学模型很好地描述。特别是，降低Ubq-UIM界面稳定性的替代确实导致了协同性的降低。
This study describes the thermodynamic characterization of a Ubq−UIM fusion construct (Ubq−UIM), designed from the ubiquitin−UIM interaction system, to determine whether it exhibits cooperativity of folding. The Ubq−UIM fusion constructs exhibit higher stability than the core Ubq molecule, consistent with the finding that the UIM helix is docked to Ubq. Temperature-induced unfolding profiles of Ubq−UIM were monitored by DSC and far-UV and near-UV CD spectroscopies. Ubq−UIM appears to exhibit cooperative unfolding as indicated by results of global fits of a two-state model to far- and near-UV CD and DSC thermal unfolding data. The cooperativity of Ubq−UIM unfolding was further tested by the amino acid substitutions that selectively stabilize or destabilize Ubq, UIM, and/or the interface. The effects of these substitutions on the thermodynamic properties of Ubq−UIM are described well by a thermodynamic model for cooperativity in proteins. In particular, a substitution that lowered the stability of the Ubq−UIM interface indeed led to a decrease in cooperativity.