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

• Applicant: Professor Dr. Daniel J. Müller
• Subject Area: Biophysics
• Term: from 2009 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 140698669

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.

• DFG Programme: Research Grants
• International Connection: Switzerland