Rajabi Khadijeh, Reuther Julia, Deuerling Elke, Radford Sheena E, Ashcroft Alison E
Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Department of Biology, University of Konstanz, 78457, Konstanz, Germany.
Protein Sci. 2015 Aug;24(8):1282-91. doi: 10.1002/pro.2702. Epub 2015 Jun 11.
The kinetics and thermodynamics of protein folding are commonly studied in vitro by denaturing/renaturing intact protein sequences. How these folding mechanisms relate to de novo folding that occurs as the nascent polypeptide emerges from the ribosome is much less well understood. Here, we have employed limited proteolysis followed by mass spectrometry analyses to compare directly free and ribosome-tethered polypeptide chains of the Src-homology 3 (SH3) domain and its unfolded variant, SH3-m10. The disordered variant was found to undergo faster proteolysis than SH3. Furthermore, the trypsin cleavage patterns observed show minor, but significant, differences for the free and ribosome-bound nascent chains, with significantly fewer tryptic peptides detected in the presence of ribosome. The results highlight the utility of limited proteolysis coupled with mass spectrometry for the structural analysis of these complex systems, and pave the way for detailed future analyses by combining this technique with chemical labeling methods (for example, hydrogen-deuterium exchange, photochemical oxidation) to analyze protein folding in real time, including in the presence of additional ribosome-associated factors.
蛋白质折叠的动力学和热力学通常在体外通过对完整蛋白质序列进行变性/复性来研究。然而，这些折叠机制与新生多肽从核糖体中出现时发生的从头折叠之间的关系却鲜为人知。在这里，我们采用了有限蛋白酶解结合质谱分析的方法，直接比较了Src同源3（SH3）结构域及其未折叠变体SH3-m10的游离和核糖体束缚多肽链。结果发现，无序变体的蛋白酶解速度比SH3更快。此外，观察到的胰蛋白酶切割模式显示，游离和核糖体结合的新生链存在微小但显著的差异，在核糖体存在的情况下检测到的胰蛋白酶肽明显减少。这些结果突出了有限蛋白酶解结合质谱分析在这些复杂系统结构分析中的实用性，并为未来通过将该技术与化学标记方法（例如，氢-氘交换、光化学氧化）相结合来实时分析蛋白质折叠，包括在存在其他核糖体相关因子的情况下进行详细分析铺平了道路。
