Duyoung Min1,2,4,5, Robert E Jefferson3,5, James U Bowie3* & Tae-Young Yoon1,2*
1National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST, Daejeon, South Korea. 2Department of Physics, KAIST, Daejeon, South Korea. 3Department of Chemistry and Biochemistry, University of California.Los Angeles, Los Angeles, California, USA. 4Present address: Department of Chemistry and Biochemistry, University of California.Los Angeles, Los Angeles, California, USA. 5These authors contributed equally to this work.
*Correspondence to : James U Bowie or Tae-Young Yoon
Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ~3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure