Silonov Sergey A, Kuklin Alexander I, Nesterov Semen V, Kuznetsova Irina M, Turoverov Konstantin K, Fonin Alexander V
Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., 194064 St. Petersburg, Russia.
Frank Laboratory of Neutron Physics (FLNP), Joint Institute for Nuclear Research, 141980 Dubna, Russia.
Int J Mol Sci. 2024 Dec 19;25(24):13600. doi: 10.3390/ijms252413600.
The 1-anilino-8-naphthalenesulfonate (ANS) fluorescent dye is widely used in protein folding studies due to the significant increase in its fluorescence quantum yield upon binding to protein hydrophobic regions that become accessible during protein unfolding. However, when modeling cellular macromolecular crowding conditions in protein folding experiments in vitro using crowding agents with guanidine hydrochloride (GdnHCl) as the denaturant, the observed changes in ANS spectral characteristics require careful consideration. This study demonstrates that crowding agents can form clusters that interact differently with ANS. Furthermore, GdnHCl can disrupt these clusters and directly affect the ANS spectral characteristics. A model for the interaction between GdnHCl, crowders, and ANS is proposed. Using bovine serum albumin (BSA) as a model protein, the limitations of using ANS for studying conformational transitions induced by GdnHCl in the presence of crowding agents are demonstrated.
1-苯胺基-8-萘磺酸盐（ANS）荧光染料因其在与蛋白质疏水区域结合时荧光量子产率显著增加而被广泛用于蛋白质折叠研究，这些疏水区域在蛋白质解折叠过程中会变得可及。然而，当在体外蛋白质折叠实验中使用盐酸胍（GdnHCl）作为变性剂的拥挤剂来模拟细胞大分子拥挤条件时，观察到的ANS光谱特征变化需要仔细考虑。本研究表明，拥挤剂可形成与ANS相互作用方式不同的聚集体。此外，GdnHCl可破坏这些聚集体并直接影响ANS光谱特征。提出了一个关于GdnHCl、拥挤剂和ANS之间相互作用的模型。以牛血清白蛋白（BSA）作为模型蛋白，证明了在存在拥挤剂的情况下使用ANS研究由GdnHCl诱导的构象转变的局限性。
