ABSTRACT 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

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INTERACTIONS DRIVE MEMBRANE PROTEIN ORGANIZATION Article Open access 30 November 2022 FREE ENERGIES OF MEMBRANE STALK FORMATION FROM A LIPIDOMICS PERSPECTIVE Article Open access 15 November

2021 SMALL-RESIDUE PACKING MOTIFS MODULATE THE STRUCTURE AND FUNCTION OF A MINIMAL DE NOVO MEMBRANE PROTEIN Article Open access 16 September 2020 REFERENCES * Engelman, D.M. et al. Membrane

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landscape of one single protein molecule. _Proc. Natl. Acad. Sci. USA_ 107, 2013–2018 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This

work was supported by the National Creative Research Initiative Program (Center for Single-Molecule Systems Biology to T.-Y.Y.) funded by the National Research Foundation of Korea and

Marine Biotechnology Program (20150220 to T.-Y.Y.) funded by the Ministry of Oceans and Fisheries of Korea, and supported by US National Institutes of Health grant 2R01GM063919 to J.U.B.

AUTHOR INFORMATION Author notes * Duyoung Min Present address: Present address: Department of Chemistry and Biochemistry, University of California–Los Angeles, Los Angeles, California, USA.,

* Duyoung Min and Robert E Jefferson: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * National Creative Research Initiative Center for Single-Molecule Systems

Biology, KAIST, Daejeon, South Korea Duyoung Min & Tae-Young Yoon * Department of Physics, KAIST, Daejeon, South Korea Duyoung Min & Tae-Young Yoon * Department of Chemistry and

Biochemistry, University of California–Los Angeles, Los Angeles, California, USA Robert E Jefferson & James U Bowie Authors * Duyoung Min View author publications You can also search for

this author inPubMed Google Scholar * Robert E Jefferson View author publications You can also search for this author inPubMed Google Scholar * James U Bowie View author publications You

can also search for this author inPubMed Google Scholar * Tae-Young Yoon View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS D.M., R.E.J.,

J.U.B. and T.-Y.Y. designed the experiments. R.E.J. expressed and purified proteins. D.M. prepared the DNA-protein hybrid sample and performed the magnetic tweezers experiments. All of the

authors analyzed the data and contributed to writing of the manuscript. CORRESPONDING AUTHORS Correspondence to James U Bowie or Tae-Young Yoon. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Results, Supplementary Figures 1–10 and Supplementary Tables 1–3.

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folding of a single membrane protein. _Nat Chem Biol_ 11, 981–987 (2015). https://doi.org/10.1038/nchembio.1939 Download citation * Received: 13 July 2015 * Accepted: 14 September 2015 *

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