Developing a new approach to characterize the self-insertion and - folding mechanisms of single membrane proteins into lipid bilayers

据报道，膜蛋白需要蛋白质传导通道的帮助才能插入并折叠到细胞膜中。然而，也有报道称，一些膜蛋白可以自发插入并折叠成膜双层。但是，是什么机制驱动蛋白质自我插入和折叠到膜中呢？哪些因素有利于和不利于这一进程？在这个项目中，我们打算建立单分子力谱（SMFS）来表征一个完全展开的多肽进入脂质膜的自我插入和折叠机制。我们已经证明SMFS足够灵敏，可以检测膜蛋白的解折叠中间体、动力学和途径。此外，我们可以表明，SMFS可以遵循部分未折叠的膜蛋白的重折叠程度和动力学。在这里，我们的目标是进一步发展这种方法，能够表征完整的自我插入和折叠的一个单一的多肽到一个功能性的膜蛋白。为了做到这一点，我们将通过它们的一个末端挑选单个膜蛋白，并将它们从膜中展开和提取出来。这种完全未折叠的肽然后将在脂质双层上移动。将跟踪多肽进入脂质膜的折叠步骤和动力学，直到最终结构被折叠。通过插入点突变，改变靶膜的脂质组成和调整缓冲液条件，我们将表征这些参数如何调节多肽的自插入和折叠成天然膜蛋白。在这个项目中要研究的膜蛋白是反向转运蛋白、细菌视紫红质和缝隙连接。然而，所开发的方法也将适用于研究其他膜蛋白的折叠过程。

It is frequently reported that membrane proteins require the assistance of protein conducting channels to insert and fold into cell membranes. However, it is also reported that some membrane proteins can spontaneously insert and fold into membrane bilayers. But which mechanisms drive this self-insertion and -folding of a protein into a membrane? Which factors favor and disfavor this process? In this project, we intend to establish single-molecule force spectroscopy (SMFS) to characterize the self-insertion and - folding mechanisms of a completely unfolded polypeptide into lipid membranes. We have demonstrated that SMFS is sensitive enough to detect unfolding intermediates, kinetics and pathways of membrane proteins. In addition, we could show that SMFS can follow the refolding extent and kinetics of a partially unfolded membrane protein. Here, we aim to further develop this approach to be able characterize the complete self-insertion and -folding of a single polypeptide into a functional membrane protein. To do so we will pick single membrane proteins by one of their terminal end and unfold and extract them from their membrane. This completely unfolded peptide will then be moved over a lipid bilayer. The folding steps and kinetics of the polypeptide into the lipid membrane will be followed until the final structure has been folded. By inserting point mutations, changing the lipid composition of the target membrane and adjusting the buffer conditions we will characterize how these parameters modulate the self-insertion and -folding of the polypeptide into the native membrane protein. Membrane proteins to be investigated in this project are antiporters, bacterial rhodopsins, and gap junctions. However, the method developed will be applicable to study the folding process of other membrane proteins as well.