Co-translational mechanisms of protein folding studied on the single molecule level

共翻译蛋白质折叠与蛋白质合成有关，受空间限制和核糖体通道内外相互作用的调节。然而，合成(即多肽链延长)和折叠是如何协同完成全功能蛋白质的，目前还不完全清楚。我们以前的工作为我们提供了必要的技术知识和经验，将基于力和荧光的单分子技术结合在一起，以实现对这种相互联系的更深层次的理解。更具体地说，我们计划重点关注三个主题。在主题一中，根据最新的文献证据，我们打算更深入地研究核糖体隧道内结合smFRET和光钳的小蛋白结构域的共翻译折叠。在第二个主题中，我们计划以共翻译的方式整合适合smFRET的荧光团，以便表征一个双域蛋白(磷酸甘油酸激酶)的不同长度的新生链，对应于合成和折叠的不同阶段。最后，在第三个主题中，我们计划更进一步，计划利用力和smFRET测量来实时观察两个结构域蛋白质的合成和折叠，并试图确定与翻译某些阶段相关的折叠事件序列。

Co-translational protein folding is coupled to protein synthesis and is mediated by spatial constraints and interactions within as well as outside the ribosomal tunnel. However, it is still not yet entirely understood how synthesis (i.e. polypeptide chain elongation) and folding cooperate to accomplish a fully functional protein. Our previous work provided us the required technical knowledge and experience to bring together force- and fluorescence-based single-molecule techniques in order to achieve a deeper understanding of this interconnection. More specifically we plan to focus on three topics. In topic one, following the latest evidence from literature, we intent to study in more depth the co-translational folding of a small protein domain inside the ribosomal tunnel combining smFRET and optical tweezers. In topic two we plan to incorporate in a co-translational manner fluorophores suitable for smFRET in order to characterize nascent chains of different length of a two-domain protein (Phosphoglycerate kinase), corresponding to different stages of synthesis and folding. Finally in topic three we aim to go one step further and plan to observe synthesis and folding of the two-domain protein in real time making use of both force and smFRET measurements and trying to identify the sequence of folding events in relation with certain stages of translation.