Shoup Daniel, Roth Andrew, Thapa Rajan, Puchalla Jason, Rye Hays S
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas.
Department of Physics, Princeton University, Princeton, New Jersey.
Biophys J. 2021 Jun 1;120(11):2192-2204. doi: 10.1016/j.bpj.2021.03.035. Epub 2021 Apr 5.
The formation and disassembly of macromolecular particles is a ubiquitous and essential feature of virtually all living organisms. Additionally, diseases are often associated with the accumulation and propagation of biologically active nanoparticles, like the formation of toxic protein aggregates in protein misfolding diseases and the growth of infectious viral particles. The heterogeneous and dynamic nature of biologically active particles can make them exceedingly challenging to study. The single-particle fluorescence technique known as burst analysis spectroscopy (BAS) was developed to facilitate real-time measurement of macromolecular particle distributions in the submicron range in a minimally perturbing, free-solution environment. Here, we develop a multicolor version of BAS and employ it to examine two problems in macromolecular assembly: 1) the extent of DNA packing heterogeneity in bacteriophage viral particles and 2) growth models of non-native protein aggregates. We show that multicolor BAS provides a powerful and flexible approach to studying hidden properties of important biological particles like viruses and protein aggregates.
大分子颗粒的形成与拆解是几乎所有生物体普遍存在且必不可少的特征。此外，疾病往往与生物活性纳米颗粒的积累和传播有关，比如在蛋白质错误折叠疾病中形成有毒蛋白质聚集体以及传染性病毒颗粒的生长。生物活性颗粒的异质性和动态特性使得对其进行研究极具挑战性。为便于在最小干扰的自由溶液环境中实时测量亚微米范围内大分子颗粒的分布，人们开发了一种名为猝发分析光谱法（BAS）的单颗粒荧光技术。在此，我们开发了一种多色版本的BAS，并将其用于研究大分子组装中的两个问题：1）噬菌体病毒颗粒中DNA包装的异质性程度；2）非天然蛋白质聚集体的生长模型。我们表明，多色BAS为研究病毒和蛋白质聚集体等重要生物颗粒的隐藏特性提供了一种强大且灵活的方法。
