Indian Institute of Science Education and Research Mohali, Knowledge City, Mohali, India.
Biophys J. 2011 Oct 5;101(7):1720-9. doi: 10.1016/j.bpj.2011.08.024.
Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine κ-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric κ-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ∼4.6 nm to ∼1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed.
天然无规或固有无序蛋白质（IDP）由于其在正常生物功能和异常蛋白质错误折叠疾病中的参与而受到强烈关注。淀粉样蛋白 IDP 的多肽链折叠被认为在蛋白质错误折叠、寡聚化和聚集导致淀粉样纤维形成中起关键作用，这与许多人类疾病有关。在这项工作中，我们使用牛 κ-酪蛋白作为淀粉样蛋白 IDP 的典型模型蛋白。使用涉及预测和光谱技术的各种生物物理工具，我们首先确定单体 κ-酪蛋白在生理条件下采用折叠前熔融球蛋白样构象集合。我们的时间分辨荧光和光散射数据表明，在链折叠时，平均流体力学半径从约 4.6nm 变化到约 1.9nm。然后，我们利用两个相隔 77 个氨基酸残基的半胱氨酸，并使用巯基反应性芘马来酰亚胺对其进行共价标记。这种双标记的蛋白质在从尿素和酸变性状态复性时，无论是在平衡还是动力学条件下，都表现出强烈的激基缔合物形成，这为生理条件下的多肽链折叠提供了有力的证据。还讨论了 IDP 链折叠在蛋白质聚集和淀粉样形成中的作用。
