Department of Physics, SUPA, University of Strathclyde, John Anderson Building, 107 Rottenrow, Glasgow G4 0NG, UK.
J Mater Chem B. 2019 Feb 21;7(7):1167-1175. doi: 10.1039/c9tb00009g. Epub 2019 Jan 31.
Protein misfolding plays a critical role in the manifestation of amyloidosis type diseases. Therefore, understanding protein unfolding and the ability to track protein unfolding in a dynamic manner are of considerable interest. Fluorescence-based techniques are powerful tools for gaining real-time information about the local environmental conditions of a probe on the nanoscale. Fluorescent gold nanoclusters (AuNCs) are a new type of fluorescent probes which are <2 nm in diameter, incredibly robust and offer highly sensitive, wavelength tuneable emission. Their small size minimises intrusion and makes AuNCs ideal for studying protein dynamics. Lysozyme has previously been used to encapsulate AuNCs. The unfolding dynamics of lysozyme under different environmental conditions have been well-studied and being an amyloid type protein makes lysozyme an ideal candidate for encapsulating AuNCs in order to test their sensitivity to protein unfolding. In this study, we tracked the fluorescence characteristics of AuNCs encapsulated in lysozyme while inducing protein unfolding using urea, sodium dodecyl sulphate (SDS) and elevated temperature and compared them to complimentary circular dichroism spectra. It is found that AuNC fluorescence emission is quenched upon induced protein unfolding either due to a decrease in Forster Resonance Energy Transfer (FRET) efficiency between tryptophan and AuNCs or solvent exposure of the AuNC. Fluorescence lifetime measurements confirmed quenching to be collisional via oxygen dissolved in a solution which increases as the AuNC was exposed to the solvent during unfolding. Moreover, the longer decay component τ was observed to decrease as the protein unfolded, due to the increased collisional quenching. It is suggested that AuNC sensitivity to solvent exposure might be utilised in the future as a new approach to studying and possibly even detecting amyloidosis type diseases.
蛋白质错误折叠在淀粉样变性疾病的表现中起着关键作用。因此，了解蛋白质的展开以及以动态方式跟踪蛋白质展开的能力是非常重要的。基于荧光的技术是获取探针在纳米尺度上局部环境条件的实时信息的有力工具。荧光金纳米团簇（AuNCs）是一种新型荧光探针，直径小于 2nm，非常坚固，提供高灵敏度、波长可调谐的发射。它们的小尺寸最小化了侵入，使 AuNCs 成为研究蛋白质动力学的理想选择。溶菌酶以前曾被用于包裹 AuNCs。溶菌酶在不同环境条件下的展开动力学已经得到了很好的研究，并且作为一种淀粉样蛋白，溶菌酶是包裹 AuNCs 的理想候选物，以测试它们对蛋白质展开的敏感性。在这项研究中，我们在使用脲、十二烷基硫酸钠（SDS）和升高温度诱导蛋白质展开的同时，跟踪了包裹在溶菌酶中的 AuNC 的荧光特性，并将其与互补圆二色性光谱进行了比较。发现 AuNC 荧光发射在诱导蛋白质展开时被猝灭，这要么是由于色氨酸和 AuNC 之间的Förster 共振能量转移（FRET）效率降低，要么是由于 AuNC 暴露于溶剂中。荧光寿命测量证实猝灭是通过溶解在溶液中的氧发生的，并且随着 AuNC 在展开过程中暴露于溶剂中，溶解氧的含量增加。此外，由于碰撞猝灭的增加，观察到较长的衰减分量 τ 随着蛋白质的展开而减小。据推测，AuNC 对溶剂暴露的敏感性可能在未来被用作研究甚至可能检测淀粉样变性疾病的新方法。
