Guliyeva Aytaj J, Gasymov Oktay K
Laboratory of Structure, Dynamics and Functions of Biomolecules, Institute of Biophysics of ANAS, 117 Z. Khalilov, Baku, AZ1171, Azerbaijan.
Biochem Biophys Rep. 2020 Nov 3;24:100843. doi: 10.1016/j.bbrep.2020.100843. eCollection 2020 Dec.
In the current study, ANS fluorescence was established as a powerful tool to study proteins in solid-state. Silk fibroin from cocoons was used as a paradigm protein. ANS incorporated into the films of silk fibroin exhibits fluorescence with two-lifetime components that can be assigned to the patches and/or cavities with distinct hydrophobicities. Decay associated spectra (DAS) of ANS fluorescence from both sites could be fit to the single log-normal component indicating their homogeneity. ANS binding sites in the protein film are specific and could be saturated by ANS titration. ANS located in the binding site that exhibits the long-lifetime fluorescence is not accessible to the water molecules and its DAS stays homogeneously broadened upon hydration of the protein film. In contrast, ANS from the sites demonstrating the short-lifetime fluorescence is accessible to water molecules. In the hydrated films, solvent-induced fluctuations produce an ensemble of binding sites with similar characters. Therefore, upon hydration, the short-lifetime DAS becomes significantly red-shifted and inhomogeneously broadened. The similar spectral features have previously been observed for ANS complexed with globular proteins in solution. The data reveal the origin of the short-lifetime fluorescence component of ANS bound to the globular proteins in aqueous solution. Findings from this study indicate that ANS is applicable to characterize dehydrated as well as hydrated protein aggregates, amyloids relevant to amyloid diseases, such as Alzheimer's, Parkinson, and prion diseases.
在本研究中，ANS荧光被确立为研究固态蛋白质的有力工具。来自蚕茧的丝素蛋白被用作典型蛋白质。掺入丝素蛋白薄膜中的ANS表现出具有两种寿命成分的荧光，这两种成分可归因于具有不同疏水性的斑块和/或空洞。来自两个位点的ANS荧光的衰减相关光谱（DAS）可以拟合为单一的对数正态成分，表明它们的同质性。蛋白质薄膜中的ANS结合位点是特异性的，并且可以通过ANS滴定饱和。位于表现出长寿命荧光的结合位点中的ANS无法被水分子接触到，并且在蛋白质薄膜水合后其DAS保持均匀展宽。相比之下，来自表现出短寿命荧光的位点的ANS可以被水分子接触到。在水合薄膜中，溶剂诱导的波动产生了具有相似特征的一系列结合位点。因此，在水合时，短寿命DAS会发生显著的红移和非均匀展宽。之前在溶液中与球状蛋白复合的ANS也观察到了类似的光谱特征。这些数据揭示了水溶液中与球状蛋白结合的ANS短寿命荧光成分的起源。本研究的结果表明，ANS适用于表征脱水以及水合的蛋白质聚集体、与淀粉样疾病（如阿尔茨海默病、帕金森病和朊病毒病）相关的淀粉样蛋白。
