Study on membrane protein folding using single-molecule magnetic tweezers

Abstract

Membrane proteins must be inserted properly into biological membranes such as endoplasmic reticulum, Golgi apparatus and plasma membrane for stable native structure and their own functioning. In terms of biogenesis, understanding how to be inserted and eventually form tertiary structure of membrane protein within the context of bilayer is essential key for elucidating membrane protein biogenesis. Although tracking the entire folding pathways of integral membrane proteins is directly breakthrough, it has remained yet to be achieved and demonstrated. This thesis demonstrates a single-molecule force spectroscopy technique, especially magnetic tweezers, that allows us to monitor the entire folding pathways of alpha-helical membrane proteins in bilayer and bicelle environments. Firstly, we completely unfold the protein at high-force above 20 pN and then lower the force below 7 pN to induce initiation of folding from a loosely stretched state where the whole transmembrane helices are aligned in a zigzag manner within the bilayer. In the zigzag state, we could impose minimal constraints on the reconstituted folding process like the circumstance of second-stage folding. By using this membrane protein folding assay, we characterized the folding pathways of two integral membrane proteins: Escherichia coli rhomboid protease GlpG and the human $\beta_{2}$-adrenergic receptor ($\beta_{2}$-AR). Albeit their enormous evolutionary distance, the folding pathways share common features. Both proteins fold in a highly ordered process starting at the N-terminus, forming in units of helical hairpins. These common features do not only suggest that the folding pathways of integral membrane proteins have evolved to maximize their fitness with co-translational insertion, but also imply that this would allow these proteins to begin folding while being translated in the biological time-scale.