Single-molecule force spectroscopy can deliver force directly to a single biomolecule (protein, DNA or their complexes) so that physical and biological information can be discovered [1][2]. Even though single-molecule force spectroscopy has been widely used for investigating physical properties of bio-molecules, folding experiments within a native membrane environment are challenging to design. We are currently applying magnetic tweezers to study membrane dynamics and membrane protein folding using native-like bicelle conditions (Figure a). When we slowly exerted force to GlpG, an Escherichia coli rhomboid protease, its six transmembrane helices showed a large unraveling in response to mechanical tension above 25 pN (Figure b). However refolding occurred at forces below 5 pN only (Figure c). Using such mechanical responses and assuming a two-state model, we could recover the folding/unfolding kinetic rates and ultimately the folding energy landscape of a single membrane protein (Figure d). We found that GlpG has only modest thermodynamic stability (ΔG= 6.5 kBT) but a large unfolding barrier (21.3 kBT), suggesting that the membrane environment allows the membrane protein to be in the folded state for extended periods of time (t1/2~3.5 h) [3].
[1] Min et al., Nat. Comm., 2013
[2] Bae et al., Nat. Comm., 2014
[3] Min et al., Nat. Chem. Biol., 2015