Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, India.
Photochem Photobiol. 2024 Jul-Aug;100(4):989-999. doi: 10.1111/php.14004. Epub 2024 Jul 19.
Various biophysical techniques have been extensively employed to study protein aggregation due to its significance. Traditionally, these methods detect aggregation at micrometer length scales and micromolar concentrations. However, unlike in vitro, protein aggregation typically occurs at nanomolar concentrations in vivo. Here, using fluorescence correlation spectroscopy (FCS), we captured bromelain aggregation at concentrations as low as ~20 nM, surpassing the detection limit of traditional methods like thioflavin T fluorescence, scattering, and fluorescence microscopy by more than one order of magnitude. Moreover, using thioflavin T fluorescence-based FCS, we have detected larger aggregates at higher bromelain concentrations, which is undetectable in FCS otherwise. Importantly, our study reveals inherent heterogeneity in bromelain aggregation, inaccessible to ensemble-averaged techniques. The presented report may provide a platform for the characterization of premature aggregates at very low protein concentrations, which are thought to be functionally significant species in protein aggregation-induced diseases.
各种生物物理技术已被广泛应用于研究蛋白质聚集，因为这具有重要意义。传统上，这些方法在微米长度尺度和微摩尔浓度下检测聚集。然而，与体外不同，蛋白质聚集通常在体内以纳摩尔浓度发生。在这里，我们使用荧光相关光谱（FCS），在低至约 20 nM 的浓度下捕获菠萝蛋白酶的聚集，比传统方法（如硫黄素 T 荧光、散射和荧光显微镜）的检测限高出一个数量级以上。此外，使用基于硫黄素 T 荧光的 FCS，我们在较高的菠萝蛋白酶浓度下检测到了更大的聚集体，而这在其他情况下是无法用 FCS 检测到的。重要的是，我们的研究揭示了菠萝蛋白酶聚集的固有异质性，这是无法通过平均技术获得的。本报告可能为在非常低的蛋白质浓度下对早期聚集物进行特征分析提供一个平台，这些早期聚集物被认为是蛋白质聚集诱导疾病中具有功能意义的物质。
