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首页> 外文期刊>Biochemistry >Identification of a Stability Determinant on the Edge of the Tet Repressor Four-Helix Bundle Dimerization Motif
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Identification of a Stability Determinant on the Edge of the Tet Repressor Four-Helix Bundle Dimerization Motif

机译：在Tet Repressor四螺旋束二聚体基元的边缘上确定稳定性的决定因素

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摘要

Isofunctional tetracycline repressor (TetR) proteins isolated from different bacteria show a sequence identity between 38 and 88% of the residues. Their active state is a homodimer formed by a four-(l-helix bundle as the main interaction motif. We utilize this sequence variation of isofunctional proteins to determine residues contributing to the stability of the four-helix bundle. The thermodynamic stabilities of two TetR proteins with 63% sequence identity were determined by urea-induced reversible jenaturation followed by fluorescence and circular dichroism. Both methods yield identical results. The Gou (H20) values are 60 and 75 kJ mol-l. We have constructed TetR hybrid proteins derived from these wild types to identify the determinant leading to the 15 kJ mol-l stability difference. Successive size reduction of the exchanged portion yielded two single residues affecting the overall protein stability. rhe P184Q exchange leads to a more stable protein, whereas the Gl81D exchange located at the solvent's ~xposed edge of the four-helix bundle is solely responsible for the reduced stability. Additional mutants )ased on crystal structures of TetR do not reveal any hint for steric interference of the Aspl81 side chain with neighboring residues. Thus, this is an example for the role played by surface-exposed turn residues For the stability of four-helix bundles. We assume that the larger conformational flexibility of Gly and the .eduction of the negative surface charge could favor formation of the .turn on the edge of the four-helix bundle.
机译：从不同细菌中分离得到的同功能四环素阻遏物（TetR）蛋白在残基的38％至88％之间显示出序列同一性。它们的活性状态是由四（l-螺旋）束作为主要相互作用基序形成的同型二聚体。我们利用同功能蛋白的这种序列变异来确定有助于四螺旋束稳定性的残基。两个TetR的热力学稳定性通过尿素诱导的可逆变色，荧光和圆二色性测定了具有63％序列同一性的蛋白，两种方法的结果相同，Gou（H20）值分别为60和75 kJ mol-l，我们构建了由这些野生型可以识别导致15 kJ mol-l稳定性差异的决定因素。交换部分的连续大小减小产生两个单个残基，影响整个蛋白质的稳定性。P184Q交换产生更稳定的蛋白质，而Gl81D交换位于溶剂在四螺旋束的并置边缘处仅是造成稳定性降低的原因。 TetR的结构没有揭示任何暗示Aspl81侧链与相邻残基的空间干扰。因此，这是表面暴露的转向残基对四螺旋束的稳定性所起的作用的一个例子。我们认为，Gly的较大构象柔韧性和负表面电荷的逸出会有利于在四螺旋束的边缘形成turn。

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