Hodges Robert S, Mills Janine, McReynolds Susanna, Kirwan J Paul, Tripet Brian, Osguthorpe David
Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, 80045, USA.
J Mol Biol. 2009 Sep 25;392(3):747-62. doi: 10.1016/j.jmb.2009.07.039. Epub 2009 Jul 21.
Nine recombinant chicken skeletal alpha-tropomyosin proteins were prepared, eight C-terminal deletion constructs and the full length protein (1-81, 1-92, 1-99, 1-105, 1-110, 1-119, 1-131, 1-260 and 1-284) and characterized by circular dichroism spectroscopy and analytical ultracentrifugation. We identified for the first time, a stability control region between residues 97 and 118. Fragments of tropomyosin lacking this region (1-81, 1-92, and 1-99) still fold into two-stranded alpha-helical coiled-coils but are significantly less stable (T(m) between 26-28.5 degrees C) than longer fragments containing this region (1-119, 1-131, 1-260 and 1-284) which show a large increase in their thermal midpoints (T(m) 40-43 degrees C) for a DeltaT(m) of 16-18 degrees C between 1-99 and 1-119. We further investigated two additional fragments that ended between residues 99 and 119, that is fragments 1-105 and 1-110. These fragments were more stable than 1-99 and less stable than 1-119, and showed that there were three separate sites that synergistically contribute to the large jump in protein stability (electrostatic clusters 97-104 and 112-118, and a hydrophobic interaction from Leu 110). All the residues involved in these stabilizing interactions are located outside the hydrophobic core a and d positions that have been shown to be the major contributor to coiled-coil stability. Our results show clearly that protein stability is more complex than previously thought and unique sites can synergistically control protein stability over long distances.
制备了9种重组鸡骨骼肌α-原肌球蛋白蛋白，包括8种C端缺失构建体和全长蛋白（1-81、1-92、1-99、1-105、1-110、1-119、1-131、1-260和1-284），并通过圆二色光谱和分析超速离心进行了表征。我们首次鉴定出97至118位残基之间的一个稳定性控制区域。缺乏该区域的原肌球蛋白片段（1-81、1-92和1-99）仍能折叠成两链α-螺旋卷曲螺旋，但稳定性明显低于包含该区域的较长片段（1-119、1-131、1-260和1-284），后者的热中点（T(m)）大幅增加（T(m)为40 - 43℃），1-99和1-119之间的ΔT(m)为16 - 18℃。我们进一步研究了另外两个在99至119位残基之间结束的片段，即片段1-105和1-110。这些片段比1-99更稳定，比1-119更不稳定，表明有三个独立的位点协同作用导致蛋白质稳定性大幅跃升（静电簇97 - 104和112 - 118，以及来自Leu 110的疏水相互作用）。参与这些稳定相互作用的所有残基都位于疏水核心a和d位置之外，而这些位置已被证明是卷曲螺旋稳定性的主要贡献者。我们的结果清楚地表明，蛋白质稳定性比以前认为的更为复杂，独特的位点可以在长距离上协同控制蛋白质稳定性。
