Sidhu Arshdeep, Segers-Nolten Ine, Subramaniam Vinod
Nanobiophysics, MESA+Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.
Nanobiophysics, MESA+Institute for Nanotechnology, University of Twente, Enschede, The Netherlands; MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands; FOM Institute AMOLF, Amsterdam, The Netherlands.
Biochim Biophys Acta. 2014 Dec;1844(12):2127-34. doi: 10.1016/j.bbapap.2014.09.007. Epub 2014 Sep 16.
The intrinsically disordered human α-synuclein (αSyn) protein exhibits considerable heterogeneity in in vitro fibrillization reactions. Using atomic force microscopy (AFM) we show that depending on the solvent conditions, A140C mutant and wild-type αSyn can be directed to reproducibly form homogeneous populations of fibrils exhibiting regular periodicity. Results from Thioflavin-T fluorescence assays, determination of residual monomer concentrations and native polyacrylamide gel electrophoresis reveal that solvent conditions including EDTA facilitate incorporation of a larger fraction of monomers into fibrils. The fibrils formed in 10mM Tris-HCl, 10mM NaCl and 0.1mM EDTA at pH7.4 display a narrow distribution of periodicities with an average value of 102±6nm for the A140C mutant and 107±9nm for wt αSyn. The ability to produce a homogeneous fibril population can be instrumental in understanding the detailed structural features of fibrils and the fibril assembly process. Moreover, the availability of morphologically well-defined fibrils will enhance the potential for use of amyloids as biological nanomaterials.
内在无序的人类α-突触核蛋白（αSyn）在体外纤维化反应中表现出相当大的异质性。使用原子力显微镜（AFM），我们发现，根据溶剂条件，A140C突变体和野生型αSyn可以被引导以可重复的方式形成具有规则周期性的均匀纤维群体。硫黄素-T荧光测定、残余单体浓度测定和天然聚丙烯酰胺凝胶电泳结果表明，包括EDTA在内的溶剂条件有助于将更大比例的单体掺入纤维中。在pH7.4的10mM Tris-HCl、10mM NaCl和0.1mM EDTA中形成的纤维显示出狭窄的周期性分布，A140C突变体的平均值为102±6nm，野生型αSyn的平均值为107±9nm。产生均匀纤维群体的能力有助于理解纤维的详细结构特征和纤维组装过程。此外，形态明确的纤维的可用性将增强淀粉样蛋白作为生物纳米材料的应用潜力。
