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The effect of boundary selection on the stability and folding of the third fibronectin type III domain from human tenascin

机译：边界选择对人腱糖蛋白上第三个III型纤连蛋白结构域稳定性和折叠的影响

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Correct selection of domain boundaries is critical for structural analysis of single domains from multimodular proteins. Folding and stability studies of the third fibronectin type III domain from human tenascin (TNfn3(1-90)) have shown that it is moderately stable (Delta G(D-N)(H2O) similar to 5 kcal mol(-1)) and folds with two-state kinetics. In an attempt to stabilize the protein, five domains were constructed with different combinations of extensions to the N- and C-termini. Thermal denaturation studies show that a specific two amino acid (Gly-Leu) extension to the C-terminus is primarily responsible for a significant increase in stability. The Delta Delta G(D-N)(H2O) of the Gly-Leu extension (TNfn3(1-92)) is 2.7 +/- 0.3 kcal mol(-1). Refolding kinetics do not differ significantly, but unfolding is slowed 40-fold. Mutation of leucine 92 to alanine does not affect stability, indicating that the stability of the extension does not come from the packing of the leucine side chain. Hydrogen exchange data suggest that the extension adds new hydrogen bonds and strengthens existing hydrogen bonds in the C-terminal interaction with the A-B and E-F loops. Removal of a very small number of hydrogen bonds substantially increases the unfolding rate, a phenomenon which may be important in stress-relaxation of FNIII-containing muscle proteins such as titin. These experiments demonstrate the importance of a small number of additional long-range interactions in the overall formation of a compact independently folding beta-sheet module. [References: 48]
机译：正确选择域边界对于多模块蛋白中单个域的结构分析至关重要。折叠和稳定性研究来自人腱糖蛋白（TNfn3（1-90））的第三个纤连蛋白III型结构域显示它是中等稳定的（Delta G（DN）（H2O）类似于5 kcal mol（-1））并折叠具有二态动力学。为了稳定蛋白质，构建了五个结构域，这些结构域具有不同的延伸至N和C末端的组合。热变性研究表明，向C端延伸的两个特定氨基酸（Gly-Leu）主要是导致稳定性显着提高的原因。 Gly-Leu延伸（TNfn3（1-92））的Delta Delta G（D-N）（H2O）为2.7 +/- 0.3 kcal mol（-1）。重折叠动力学没有显着差异，但展开速度减慢了40倍。亮氨酸92突变为丙氨酸不会影响稳定性，表明延伸的稳定性不是来自亮氨酸侧链的堆积。氢交换数据表明，该延伸增加了新的氢键，并在与A-B和E-F环的C末端相互作用中增强了现有的氢键。除去非常少的氢键会大大提高解折叠速率，这种现象在应力释放含FNIII的肌蛋白（如肌动蛋白）中可能很重要。这些实验证明了在紧凑独立折叠的β-sheet模块的整体形成过程中，少量其他远程相互作用的重要性。 [参考：48]

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