Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG

Nature Chemical Biology volume 12, pages 353–360 (2016)Cite this article

Subjects

Abstract

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Principle of steric trapping and steric trapping probes developed in this study.
Figure 2: GlpG reversibly unfolds by double binding of mSA.
Figure 3: DEER suggests steric trapping induce wide separation of two biotinylated sites.
Figure 4: Thermodynamic stability of GlpG using steric trapping and SDS denaturation.
Figure 5: Dependence of thermodynamic stability of GlpG on SDS mole fraction.
Figure 6: Cooperativity map reveals a network of clustered cooperative and localized interactions for the stability of GlpG under a native micellar condition.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Bryngelson, J.D., Onuchic, J.N., Socci, N.D. & Wolynes, P.G. Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins 21, 167–195 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Oliveberg, M. & Wolynes, P.G. The experimental survey of protein-folding energy landscapes. Q. Rev. Biophys. 38, 245–288 (2005).

    CAS  PubMed  Google Scholar 

  3. Dill, K.A. & Chan, H.S. From Levinthal to pathways to funnels. Nat. Struct. Biol. 4, 10–19 (1997).

    CAS  PubMed  Google Scholar 

  4. Chamberlain, A.K., Handel, T.M. & Marqusee, S. Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH. Nat. Struct. Biol. 3, 782–787 (1996).

    CAS  PubMed  Google Scholar 

  5. Bai, Y., Sosnick, T.R., Mayne, L. & Englander, S.W. Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Sekhar, A. & Kay, L.E. NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. Proc. Natl. Acad. Sci. USA 110, 12867–12874 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Park, C. & Marqusee, S. Probing the high energy states in proteins by proteolysis. J. Mol. Biol. 343, 1467–1476 (2004).

    CAS  PubMed  Google Scholar 

  8. Sharon, M. & Robinson, C.V. The role of mass spectrometry in structure elucidation of dynamic protein complexes. Annu. Rev. Biochem. 76, 167–193 (2007).

    CAS  PubMed  Google Scholar 

  9. Bai, Y. & Englander, S.W. Future directions in folding: the multi-state nature of protein structure. Proteins 24, 145–151 (1996).

    CAS  PubMed  Google Scholar 

  10. Cui, Q. & Karplus, M. Allostery and cooperativity revisited. Protein Sci. 17, 1295–1307 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Nussinov, R. & Tsai, C.J. Allostery in disease and in drug discovery. Cell 153, 293–305 (2013).

    CAS  PubMed  Google Scholar 

  12. le Coutre, J., Kaback, H.R., Patel, C.K., Heginbotham, L. & Miller, C. Fourier transform infrared spectroscopy reveals a rigid alpha-helical assembly for the tetrameric Streptomyces lividans K+ channel. Proc. Natl. Acad. Sci. USA 95, 6114–6117 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Joh, N.H. et al. Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins. Nature 453, 1266–1270 (2008).

    CAS  PubMed  Google Scholar 

  14. Blois, T.M., Hong, H., Kim, T.H. & Bowie, J.U. Protein unfolding with a steric trap. J. Am. Chem. Soc. 131, 13914–13915 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Chang, Y.C. & Bowie, J.U. Measuring membrane protein stability under native conditions. Proc. Natl. Acad. Sci. USA 111, 219–224 (2014).

    CAS  PubMed  Google Scholar 

  16. Hong, H., Blois, T.M., Cao, Z. & Bowie, J.U. Method to measure strong protein-protein interactions in lipid bilayers using a steric trap. Proc. Natl. Acad. Sci. USA 107, 19802–19807 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Hong, H. & Bowie, J.U. Dramatic destabilization of transmembrane helix interactions by features of natural membrane environments. J. Am. Chem. Soc. 133, 11389–11398 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Jefferson, R.E., Blois, T.M. & Bowie, J.U. Membrane proteins can have high kinetic stability. J. Am. Chem. Soc. 135, 15183–15190 (2013).

    CAS  PubMed  Google Scholar 

  19. Lee, J.R., Urban, S., Garvey, C.F. & Freeman, M. Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila. Cell 107, 161–171 (2001).

    CAS  PubMed  Google Scholar 

  20. McQuibban, G.A., Saurya, S. & Freeman, M. Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541 (2003).

    CAS  PubMed  Google Scholar 

  21. Stevenson, L.G. et al. Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase. Proc. Natl. Acad. Sci. USA 104, 1003–1008 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Urban, S., Lee, J.R. & Freeman, M. Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107, 173–182 (2001).

    CAS  PubMed  Google Scholar 

  23. Baker, R.P. & Urban, S. Architectural and thermodynamic principles underlying intramembrane protease function. Nat. Chem. Biol. 8, 759–768 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Paslawski, W. et al. Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops. Proc. Natl. Acad. Sci. USA 112, 7978–7983 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Min, D., Jefferson, R.E., Bowie, J.U. & Yoon, T.Y. Mapping the energy landscape for second-stage folding of a single membrane protein. Nat. Chem. Biol. 11, 981–987 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Vosyka, O. et al. Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay. Proc. Natl. Acad. Sci. USA 110, 2472–2477 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Zoll, S. et al. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures. EMBO J. 33, 2408–2421 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Sherratt, A.R., Blais, D.R., Ghasriani, H., Pezacki, J.P. & Goto, N.K. Activity-based protein profiling of the Escherichia coli GlpG rhomboid protein delineates the catalytic core. Biochemistry 51, 7794–7803 (2012).

    CAS  PubMed  Google Scholar 

  29. Arutyunova, E. et al. Allosteric regulation of rhomboid intramembrane proteolysis. EMBO J. 33, 1869–1881 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Howarth, M. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat. Methods 3, 267–273 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Klumb, L.A., Chu, V. & Stayton, P.S. Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex. Biochemistry 37, 7657–7663 (1998).

    CAS  PubMed  Google Scholar 

  32. Jeschke, G. DEER distance measurements on proteins. Annu. Rev. Phys. Chem. 63, 419–446 (2012).

    CAS  PubMed  Google Scholar 

  33. Dockter, C. et al. Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR. Proc. Natl. Acad. Sci. USA 106, 18485–18490 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Krishnamani, V., Hegde, B.G., Langen, R. & Lanyi, J.K. Secondary and tertiary structure of bacteriorhodopsin in the SDS denatured state. Biochemistry 51, 1051–1060 (2012).

    CAS  PubMed  Google Scholar 

  35. Hubbell, W.L., Cafiso, D.S. & Altenbach, C. Identifying conformational changes with site-directed spin labeling. Nat. Struct. Biol. 7, 735–739 (2000).

    CAS  PubMed  Google Scholar 

  36. Mok, Y.K., Kay, C.M., Kay, L.E. & Forman-Kay, J. NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions. J. Mol. Biol. 289, 619–638 (1999).

    CAS  PubMed  Google Scholar 

  37. Byler, D.M. & Susi, H. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers 25, 469–487 (1986).

    CAS  PubMed  Google Scholar 

  38. Myers, J.K., Pace, C.N. & Scholtz, J.M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138–2148 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Renthal, R. An unfolding story of helical transmembrane proteins. Biochemistry 45, 14559–14566 (2006).

    CAS  PubMed  Google Scholar 

  40. Otzen, D.E. Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein. J. Mol. Biol. 330, 641–649 (2003).

    CAS  PubMed  Google Scholar 

  41. Bédard, S., Mayne, L.C., Peterson, R.W., Wand, A.J. & Englander, S.W. The foldon substructure of staphylococcal nuclease. J. Mol. Biol. 376, 1142–1154 (2008).

    PubMed  Google Scholar 

  42. Llinás, M., Gillespie, B., Dahlquist, F.W. & Marqusee, S. The energetics of T4 lysozyme reveal a hierarchy of conformations. Nat. Struct. Biol. 6, 1072–1078 (1999).

    PubMed  Google Scholar 

  43. Dutta, A. et al. Characterization of membrane protein non-native states. 2. The SDS-unfolded states of rhodopsin. Biochemistry 49, 6329–6340 (2010).

    CAS  PubMed  Google Scholar 

  44. Lemberg, M.K. & Freeman, M. Functional and evolutionary implications of enhanced genomic analysis of rhomboid intramembrane proteases. Genome Res. 17, 1634–1646 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Dill, K.A. & Shortle, D. Denatured states of proteins. Annu. Rev. Biochem. 60, 795–825 (1991).

    CAS  PubMed  Google Scholar 

  46. Xue, Y. & Ha, Y. Large lateral movement of transmembrane helix S5 is not required for substrate access to the active site of rhomboid intramembrane protease. J. Biol. Chem. 288, 16645–16654 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Wu, Z. et al. Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry. Nat. Struct. Mol. Biol. 13, 1084–1091 (2006).

    CAS  PubMed  Google Scholar 

  48. Liu, T., Whitten, S.T. & Hilser, V.J. Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble. Proc. Natl. Acad. Sci. USA 104, 4347–4352 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang, Y., Maegawa, S., Akiyama, Y. & Ha, Y. The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG. J. Mol. Biol. 374, 1104–1113 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Akiyama, Y. & Maegawa, S. Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease. Mol. Microbiol. 64, 1028–1037 (2007).

    CAS  PubMed  Google Scholar 

  51. Hong, H., Chang, Y.C. & Bowie, J.U. Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping. Methods Mol. Biol. 1063, 37–56 (2013).

    PubMed  PubMed Central  Google Scholar 

  52. Bolen, D.W. & Santoro, M.M. Unfolding free energy changes determined by the linear extrapolation method. 2. Incorporation of delta G degrees N-U values in a thermodynamic cycle. Biochemistry 27, 8069–8074 (1988).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by startup funds from Michigan State University (H.H.), a Hunt for a Cure grant for basic cystic fibrosis research (H.H.), US National Cancer Institute R01CA149451 (X.H.), US National Eye Institute R01EY005216 (W.L.H.) and the Jules Stein Professor endowment (W.L.H.). The authors thank J. McCracken for general advice on electron paramagnetic resonance (EPR) and for helping with continuous-wave EPR measurements and C. Altenbach for the DEER analysis program. We thank E. Deans and C. Arthur for helping with protein preparations. We also thank R. Jefferson, N. Woodall, Y.-C. Chang, D. Weliky, L. Kroos, J. McCracken and J. Bowie for critical reading of the manuscript. We appreciate suggestions from the anonymous reviewers, which greatly helped improving the quality of this manuscript.

Author information

Author notes

  1. Zhongyu Yang

    Present address: Present address: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota, USA.,

  2. Ruiqiong Guo and Kristen Gaffney: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Michigan State University, East Lansing, Michigan, USA

    Ruiqiong Guo, Miyeon Kim, Suttipun Sungsuwan, Xuefei Huang & Heedeok Hong

  2. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA

    Kristen Gaffney & Heedeok Hong

  3. Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, California, USA

    Zhongyu Yang & Wayne L Hubbell

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

    Zhongyu Yang & Wayne L Hubbell

Authors
  1. Ruiqiong Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kristen Gaffney
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhongyu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miyeon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Suttipun Sungsuwan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xuefei Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wayne L Hubbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Heedeok Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.G., K.G., M.K. and H.H. designed experiments, expressed and purified proteins, and performed steric trapping and other biochemical experiments; R.G., X.H. and H.H. designed new steric trapping probes; R.G., S.S. and X.H. synthesized and characterized the new probes; Z.Y. and M.K. performed EPR measurements; Z.Y., M.K., H.H. and W.L.H. interpreted DEER data; All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Heedeok Hong.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1, Supplementary Figures 1–14 and Supplementary Note (Synthetic Procedures). (PDF 3217 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guo, R., Gaffney, K., Yang, Z. et al. Steric trapping reveals a cooperativity network in the intramembrane protease GlpG. Nat Chem Biol 12, 353–360 (2016). https://doi.org/10.1038/nchembio.2048

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2048

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing