使用同位素标记的小角中子散射法测定溶液中淀粉样纤维的伸长率和平均长度
Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering.
作者信息
Eves Ben J, Doutch James J, Terry Ann E, Yin Han, Moulin Martine, Haertlein Michael, Forsyth V Trevor, Flagmeier Patrick, Knowles Tuomas P J, Dias David M, Lotze Gudrun, Seddon Annela M, Squires Adam M
机构信息
Department of Chemistry, University of Bath Bath UK
ISIS Facility, Rutherford Appleton Laboratory, STFC, Chilton Didcot OX11 0QX UK.
出版信息
RSC Chem Biol. 2021 Apr 14;2(4):1232-1238. doi: 10.1039/d1cb00001b. eCollection 2021 Aug 5.
We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate. In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique. Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg ml seeds in 2.5 mg ml deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min, and an average seed length estimate of 4.2 ± 1.3 μm.
我们展示了一种求解方法,该方法能够估计组装过程中淀粉样纤维的伸长率和平均纤维长度。该方法包括在种子生长的初始阶段获取实时中子散射数据,使用对比度匹配缓冲液使种子对中子有效不可见。随着氘代单体添加到种子上,标记的生长末端产生散射图案,我们将其建模为圆柱体,其长度随时间的增加给出伸长率。此外,信号的绝对强度可用于确定单位体积内生长末端的数量,进而提供种子长度的估计值。在伸长过程中,末端数量没有显著变化,这表明与在预先存在的纤维末端生长相比,任何自发或二次成核都不显著,此外还为该技术提供了一种内部验证方法。我们在室温下使用2.5 mg/ml氘代单体中的1.2 mg/ml种子对α-突触核蛋白纤维的初始生长进行的实验,得到的伸长率为6.3±0.5 Å/min,平均种子长度估计值为4.2±1.3 μm。