Binding mode of Thioflavin T in insulin amyloid fibrils
Binding mode of Thioflavin T in insulin amyloid fibrils
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DOI：
10.1016/j.jsb.2007.06.004
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发表时间：
2007-09-01
影响因子：
3
通讯作者：
Frokjaer, Sven
中科院分区：
文献类型：
--
作者：
Groenning, Minna;Norrman, Mathias;Frokjaer, Sven
淀粉样原纤维具有各种共同的结构特征，并且它们的存在可以通过硫磺素T（ThT）检测。本文研究了ThT与胰岛素淀粉样纤维的结合方式。Scatchard分析和等温滴定量热法（ITC）表明，至少有两个结合位点的人口。具有最强结合的结合位点群体负责特征性ThT荧光。这种结合具有每摩尔原纤维形式的胰岛素结合约0.1摩尔ThT的能力。结合能力不受pH值的影响，但在低pH值的亲和力是最低的。值得注意的是，存在的第三个结合过程之前，其他过程的建议由ITC。根据X射线衍射图，ThT的结合仅导致原纤维结构的微小变化，其中观察到相对于IIA的片间距离在16 A处略微更占优势的赤道反射。没有观察到4.8埃的链间距离的变化。根据我们的研究结果，我们提出ThT结合在平行于原纤维轴的空腔中，例如，形成原纤维的原丝之间先前已经在胰岛素原纤维和几种其他淀粉样原纤维模型中提出了这样的空腔。(c)2007年爱思唯尔公司All rights reserved.
Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examined. Scatchard analysis and isothermal titration calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the X-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16 A relative to the intersheet distance of I I A was observed. No change in the interstrand distance of 4.8 angstrom was observed. On the basis of our results, we propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models. (c) 2007 Elsevier Inc. All rights reserved.