Analysis of the Native Structure, Stability and Aggregation of Biotinylated Human Lysozyme
Analysis of the Native Structure, Stability and Aggregation of Biotinylated Human Lysozyme
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DOI：
10.1371/journal.pone.0050192
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发表时间：
2012-11-16
期刊：
影响因子：
3.7
通讯作者：
Kumita, Janet R.
中科院分区：
文献类型：
--
作者：
Ahn, Minkoo;De Genst, Erwin;Kumita, Janet R.
人类溶菌酶的突变变异形成的纤维与一种致命的遗传性非神经性系统性淀粉样变性有关。确定溶菌酶聚集的机制细节对于了解该病的起源和发展以及相关的错误折叠情况至关重要。在这项研究中，我们证明了生物素部分可以在人溶菌酶的Lys33位点特异性地引入。我们使用生物物理技术证明，这种修饰不会明显改变蛋白质天然状态的结构和稳定性，形成淀粉样纤维的能力也没有变化。通过利用生物素-亲和素的相互作用，我们证明了超分辨率荧光显微镜可以产生成熟纤维的详细图像。这种方法可以很容易地将额外的探针引入蛋白质中，从而提供了一种手段，通过这些手段可以详细地了解溶菌酶及其变体在各种条件下的聚集过程的性质。
Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions.