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Mapping the energy landscape for second-stage folding of a single membrane protein

Nature Chemical Biology volume 11, pages 981–987 (2015)Cite this article

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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 (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.

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Figure 1: Cooperative unfolding and refolding of GlpG in bicelles.
Figure 2: C- to N-terminus unfolding of single GlpG with two intermediates.
Figure 3: Folding energy landscape of GlpG.
Figure 4: Comparison of kinetic and thermodynamic properties between WT and mutant GlpG.
Figure 5: How the folding energy landscape of GlpG may prevent dangerous misfolded states.

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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.

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Author notes

  1. Duyoung Min

    Present address: Present address: Department of Chemistry and Biochemistry, University of California–Los Angeles, Los Angeles, California, USA.,

  2. Duyoung Min and Robert E Jefferson: These authors contributed equally to this work.

Authors and Affiliations

  1. National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST, Daejeon, South Korea

    Duyoung Min & Tae-Young Yoon

  2. Department of Physics, KAIST, Daejeon, South Korea

    Duyoung Min & Tae-Young Yoon

  3. Department of Chemistry and Biochemistry, University of California–Los Angeles, Los Angeles, California, USA

    Robert E Jefferson & James U Bowie

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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.

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Min, D., Jefferson, R., Bowie, J. et al. Mapping the energy landscape for second-stage folding of a single membrane protein. Nat Chem Biol 11, 981–987 (2015). https://doi.org/10.1038/nchembio.1939

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