Protein molecules typically unfold (denature) when subjected to extremes of heat, cold, pH, solvent composition, or mechanical stress. One might expect that shearing forces induced by a nonuniform fluid flow would also destabilize proteins, as when a protein solution flows rapidly through a narrow channel. However, although the protein literature contains many references to shear denaturation, we find little quantitative evidence for the phenomenon. We have investigated whether a high shear can destabilize a small globular protein to any measurable extent. We study a protein (horse cytochrome c, 104 amino acids) whose fluorescence increases sharply upon unfolding. By forcing the sample through a silica capillary (inner diameter 150–180 μm) at speeds approaching 10 m/s, we subject the protein to shear rates dvz/dr as large as ∼2 × 105 s−1 while illuminating it with an ultraviolet laser. We can readily detect fluorescence changes of <1%, corresponding to shifts of <∼0.01 kJ/mol in the stability of the folded state. We find no evidence that even our highest shear rates significantly destabilize the folded protein. A simple model suggests that extraordinary shear rates, ∼107 s−1, would be required to denature typical small, globular proteins in water.
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机译:当受到极端的热,冷,pH,溶剂组成或机械应力时,蛋白质分子通常会展开(变性)。人们可能会期望由不均匀的流体流动引起的剪切力也会破坏蛋白质的稳定性,就像蛋白质溶液在狭窄的通道中快速流动一样。然而,尽管蛋白质文献中有许多关于剪切变性的参考文献,但我们几乎没有定量证据证明这种现象。我们已经研究了高剪切是否可以在任何可测量的范围内破坏小球状蛋白的稳定性。我们研究了一种蛋白质(马细胞色素c,104个氨基酸),其荧光在展开时会急剧增加。通过以接近10 m / s的速度迫使样品通过硅胶毛细管(内径150–180μm),我们使蛋白质的剪切速率dvz / dr大至约2×10 5 sup> s -1 sup>并用紫外线照射。我们可以很容易地检测到<1%的荧光变化,对应于折叠状态稳定性下<〜0.01 kJ / mol的变化。我们没有证据表明,即使我们的最高剪切速率也会显着破坏折叠蛋白的稳定性。一个简单的模型表明,要使水中的典型小球形蛋白质变性,就需要非凡的剪切速率,约10 7 sup> s -1 sup>。
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