State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China.
State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China; Key Laboratory of Bio-Resources and Eco-Environment of MOE, College of Life Science, Sichuan University, Chengdu, China.
Biophys J. 2012 Aug 8;103(3):558-566. doi: 10.1016/j.bpj.2012.07.002.
Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.
分子内伴侣（IMC）是蛋白质一级结构中编码的特定结构域/片段，对同一分子中其他结构域的聚集具有伴侣样活性。在这项研究中，我们发现连接肽的截断大大促进了兔肌肉肌酸激酶（RMCK）的分离 C 末端结构域（CTD）的热聚集。连接肽的存在（共价连接到 CTD）或额外提供合成的连接肽都可以成功地以浓度依赖的方式保护 RMCK 的 CTD 免受聚集。连接肽的截断片段也表现出类似伴侣的效应，但效率较低，这表明其 C 末端一半在连接肽的 IMC 功能中很重要。通过分子动力学模拟鉴定了 RMCK 的 CTD 中的聚集部位。三个关键疏水性残基的突变分析对具有完整或部分连接肽的 CTD 的热聚集产生了相反的影响，证实了连接肽作为盖子的作用，可防止疏水性残基暴露于溶剂中。这些观察结果表明，多结构域蛋白中的连接肽可以作为 IMC 促进易于聚集的结构域的正确折叠。此外，蛋白水解后 IMC 连接肽的完整性调节了非通路聚集物的产生，这对于由聚集蛋白水解片段的毒性作用引起的一些疾病的发生可能很重要。
