Laboratory of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, Leninsky pr. 33, Moscow, 119071, Russia.
Laboratory of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, Leninsky pr. 33, Moscow, 119071, Russia.
Biochem Biophys Res Commun. 2019 Jan 22;508(4):1101-1105. doi: 10.1016/j.bbrc.2018.12.015. Epub 2018 Dec 11.
Small heat shock proteins (sHsps) are molecular chaperones preventing protein aggregation. Dynamics of quaternary structure plays an important role in the chaperone-like activity of sHsps. However, an interrelation between the oligomeric state and chaperone-like activity of sHsps remains insufficiently characterized. Most of the accumulated data were obtained in dilute protein solutions, leaving the question of the oligomeric state of sHsps in crowded intracellular media largely unanswered. Here, we analyzed the effect of crowding on the oligomeric state of αB-crystallin (αB-Cr) using analytical ultracentrifugation. Marked increase in the sedimentation coefficient of αB-Cr was observed in the presence of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and trimethylamine N-oxide (TMAO) at 48 °C. An especially pronounced effect was detected for the PEG and TMAO mixture, where the sedimentation coefficient (s) of αB-Cr increased from 10.7 S in dilute solution up to 40.7 S in the presence of crowding agents. In the PEG + TMAO mixture, addition of model protein substrate (muscle glycogen phosphorylase b) induced dissociation of large αB-Cr oligomers and formation of complexes with smaller sedimentation coefficients, supporting the idea that, under crowding conditions, protein substrates can promote dissociation of large αB-Cr oligomers.
小分子热休克蛋白（sHsps）是一种分子伴侣，可防止蛋白质聚集。四级结构的动态变化在 sHsps 的伴侣样活性中起着重要作用。然而，寡聚状态和 sHsps 的伴侣样活性之间的关系仍未得到充分表征。大多数已积累的数据是在稀蛋白溶液中获得的，这使得 sHsps 在拥挤的细胞内介质中的寡聚状态问题很大程度上未得到解答。在这里，我们使用分析超速离心法分析了拥挤对αB-晶体蛋白（αB-Cr）寡聚状态的影响。在 48°C 时，在聚乙二醇（PEG）、聚乙烯吡咯烷酮（PVP）和三甲基氧化胺（TMAO）存在的情况下，αB-Cr 的沉降系数明显增加。在 PEG 和 TMAO 的混合物中，检测到特别明显的效果，其中 αB-Cr 的沉降系数（s）从稀溶液中的 10.7 S 增加到存在拥挤剂时的 40.7 S。在 PEG+TMAO 混合物中，添加模型蛋白底物（肌肉糖原磷酸化酶 b）诱导大的αB-Cr 寡聚物的解离，并形成具有较小沉降系数的复合物，这支持了这样的观点，即在拥挤条件下，蛋白底物可以促进大的αB-Cr 寡聚物的解离。
