Skip to main content
SpringerLink
Log in

Time Window Expansion for HDX Analysis of an Intrinsically Disordered Protein

  • Research Article
  • Published:
Journal of The American Society for Mass Spectrometry

Abstract

Application of typical HDX methods to examine intrinsically disordered proteins (IDP), proteins that are natively unstructured and highly dynamic at physiological pH, is limited because of the rapid exchange of unprotected amide hydrogens with solvent. The exchange rates of these fast exchanging amides are usually faster than the shortest time scale (10 s) employed in typical automated HDX-MS experiments. Considering the functional importance of IDPs and their association with many diseases, it is valuable to develop methods that allow the study of solution dynamics of these proteins as well as the ability to probe the interaction of IDPs with their wide range of binding partners. Here, we report the application of time window expansion to the millisecond range by altering the on-exchange pH of the HDX experiment to study a well-characterized IDP; the activation domain of the nuclear receptor coactivator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). This method enabled mapping the regions of PGC-1α that are stabilized upon binding the ligand binding domain (LBD) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). We further demonstrate the method’s applicability to other binding partners of the IDP PGC-1α and pave the way for characterizing many other biologically important ID proteins.

ᇵ

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Dunker, A.K., Lawson, J.D., Brown, C.J., Williams, R.M., Romero, P., Oh, J.S., Oldfield, C.J., Campen, A.M., Ratliff, C.M., Hipps, K.W., Ausio, J., Nissen, M.S., Reeves, R., Kang, C., Kissinger, C.R., Bailey, R.W., Griswold, M.D., Chiu, W., Garner, E.C., Obradovic, Z.: Intrinsically disordered protein. J. Mol. Graph. Model. 19, 26–59 (2001)

    Google Scholar 

  2. Sigler, P.B.: Transcriptional activation. Acid blobs and negative noodles. Nature 333, 210–212 (1988)

    Article  CAS  Google Scholar 

  3. Wright, P.E., Dyson, H.J.: Intrinsically unstructured proteins: reassessing the protein structure-function paradigm. J. Mol. Biol. 293, 321–331 (1999)

    Article  CAS  Google Scholar 

  4. Uversky, V.N., Gillespie, J.R., Fink, A.L.: Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins 41, 415–427 (2000)

    Article  CAS  Google Scholar 

  5. Uversky, V.N., Oldfield, C.J., Dunker, A.K.: Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu. Rev. Biophys. 37, 215–246 (2008)

    Article  CAS  Google Scholar 

  6. Romero, P., Obradovic, Z., Li, X., Garner, E.C., Brown, C.J., Dunker, A.K.: Sequence complexity of disordered protein. Proteins 42, 38–48 (2001)

    Article  CAS  Google Scholar 

  7. Ward, J.J., Sodhi, J.S., McGuffin, L.J., Buxton, B.F., Jones, D.T.: Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J. Mol. Biol. 337, 635–645 (2004)

    Article  CAS  Google Scholar 

  8. Weathers, E.A., Paulaitis, M.E., Woolf, T.B., Hoh, J.H.: Reduced amino acid alphabet is sufficient to accurately recognize intrinsically disordered protein. FEBS Lett. 576, 348–352 (2004)

    Article  CAS  Google Scholar 

  9. Sickmeier, M., Hamilton, J.A., LeGall, T., Vacic, V., Cortese, M.S., Tantos, A., Szabo, B., Tompa, P., Chen, J., Uversky, V.N., Obradovic, Z., Dunker, A.K.: DisProt: the database of disordered proteins. Nucleic Acids Res. 35, D786–D793 (2007)

    Google Scholar 

  10. Dosztanyi, Z., Csizmok, V., Tompa, P., Simon, I.: IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005)

    Article  CAS  Google Scholar 

  11. Dunker, A.K., Brown, C.J., Lawson, J.D., Iakoucheva, L.M., Obradovic, Z.: Intrinsic disorder and protein function. Biochemistry 41, 6573–6582 (2002)

    Article  CAS  Google Scholar 

  12. Oldfield, C.J., Cheng, Y., Cortese, M.S., Brown, C.J., Uversky, V.N., Dunker, A.K.: Comparing and combining predictors of mostly disordered proteins. Biochemistry 44, 1989–2000 (2005)

    Article  CAS  Google Scholar 

  13. Dyson, H.J., Wright, P.E.: Coupling of folding and binding for unstructured proteins. Curr. Opin. Struct. Biol. 12, 54–60 (2002)

    Article  CAS  Google Scholar 

  14. Lacy, E.R., Filippov, I., Lewis, W.S., Otieno, S., Xiao, L., Weiss, S., Hengst, L., Kriwacki, R.W.: p27 Binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding. Nat. Struct. Mol. Biol. 11, 358–364 (2004)

    Article  CAS  Google Scholar 

  15. Xie, H., Vucetic, S., Iakoucheva, L.M., Oldfield, C.J., Dunker, A.K., Obradovic, Z., Uversky, V.N.: Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins. J. Proteome Res. 6, 1917–1932 (2007)

    Article  CAS  Google Scholar 

  16. Weinreb, P.H., Zhen, W., Poon, A.W., Conway, K.A., Lansbury Jr., P.T.: NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry 35, 13709–13715 (1996)

    Article  CAS  Google Scholar 

  17. Eliezer, D.: Biophysical characterization of intrinsically disordered proteins. Curr. Opin. Struct. Biol. 19, 23–30 (2009)

    Article  CAS  Google Scholar 

  18. Wright, P.E., Dyson, H.J.: Linking folding and binding. Curr. Opin. Struct. Biol. 19, 31–38 (2009)

    Article  CAS  Google Scholar 

  19. Zhang, X., Chien, E.Y., Chalmers, M.J., Pascal, B.D., Gatchalian, J., Stevens, R.C., Griffin, P.R.: Dynamics of the beta2-adrenergic G-protein coupled receptor revealed by hydrogen–deuterium exchange. Anal. Chem. 82, 1100–1108 (2010)

    Article  CAS  Google Scholar 

  20. Zhang, J., Chalmers, M.J., Stayrook, K.R., Burris, L.L., Garcia-Ordonez, R.D., Pascal, B.D., Burris, T.P., Dodge, J.A., Griffin, P.R.: Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer. Structure 18, 1332–1341 (2010)

    Article  CAS  Google Scholar 

  21. West, G.M., Chien, E.Y., Katritch, V., Gatchalian, J., Chalmers, M.J., Stevens, R.C., Griffin, P.R.: Ligand-dependent perturbation of the conformational ensemble for the GPCR beta2 adrenergic receptor revealed by HDX. Structure 19, 1424–1432 (2011)

    Article  CAS  Google Scholar 

  22. Chalmers, M.J., Busby, S.A., Pascal, B.D., West, G.M., Griffin, P.R.: Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions. Expert Rev. Proteom. 8, 43–59 (2011)

    Article  CAS  Google Scholar 

  23. Englander, S.W.: Hydrogen exchange and mass spectrometry: A historical perspective. J. Am. Soc. Mass Spectrom. 17, 1481–1489 (2006)

    Article  CAS  Google Scholar 

  24. Coales, S.J., Sook Yen, E., Lee, J.E., Ma, A., Morrow, J.A., Hamuro, Y.: Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions. Rapid Commun. Mass Spectrom. 24, 3585–3592 (2010)

    Article  CAS  Google Scholar 

  25. Sharma, S., Zheng, H., Huang, Y.J., Ertekin, A., Hamuro, Y., Rossi, P., Tejero, R., Acton, T.B., Xiao, R., Jiang, M., Zhao, L., Ma, L.C., Swapna, G.V., Aramini, J.M., Montelione, G.T.: Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry. Proteins 76, 882–894 (2009)

    Google Scholar 

  26. Pantazatos, D., Kim, J.S., Klock, H.E., Stevens, R.C., Wilson, I.A., Lesley, S.A., Woods Jr., V.L.: Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS. Proc. Natl. Acad. Sci. U. S. A. 101, 751–756 (2004)

    Article  CAS  Google Scholar 

  27. Mitchell, J.L., Trible, R.P., Emert-Sedlak, L.A., Weis, D.D., Lerner, E.C., Applen, J.J., Sefton, B.M., Smithgall, T.E., Engen, J.R.: Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein. J. Mol. Biol. 366, 1282–1293 (2007)

    Article  CAS  Google Scholar 

  28. Hansen, J.C., Wexler, B.B., Rogers, D.J., Hite, K.C., Panchenko, T., Ajith, S., Black, B.E.: DNA binding restricts the intrinsic conformational flexibility of methyl CpG binding protein 2 (MeCP2). J. Biol. Chem. 286, 18938–18948 (2011)

    Article  CAS  Google Scholar 

  29. Keppel, T.R., Howard, B.A., Weis, D.D.: Mapping unstructured regions and synergistic folding in intrinsically disordered proteins with amide H/D exchange mass spectrometry. Biochemistry 50, 8722–8732 (2011)

    Article  CAS  Google Scholar 

  30. Simmons, D.A., Konermann, L.: Characterization of transient protein folding intermediates during myoglobin reconstitution by time-resolved electrospray mass spectrometry with on-line isotopic pulse labeling. Biochemistry 41, 1906–1914 (2002)

    Article  CAS  Google Scholar 

  31. Rist, W., Rodriguez, F., Jorgensen, T.J., Mayer, M.P.: Analysis of subsecond protein dynamics by amide hydrogen exchange and mass spectrometry using a quenched-flow setup. Protein Sci. 14, 626–632 (2005)

    Article  CAS  Google Scholar 

  32. Lin, J., Handschin, C., Spiegelman, B.M.: Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab. 1, 361–370 (2005)

    Article  Google Scholar 

  33. Burris, T.P., Busby, S.A., Griffin, P.R.: Targeting orphan nuclear receptors for treatment of metabolic diseases and autoimmunity. Chem. Biol. 19, 51–59 (2012)

    Article  CAS  Google Scholar 

  34. Deblois, G., Giguere, V.: Functional and physiological genomics of estrogen-related receptors (ERRs) in health and disease. Biochim. Biophys. Acta 1812, 1032–1040 (2011)

    Article  CAS  Google Scholar 

  35. Devarakonda, S., Gupta, K., Chalmers, M.J., Hunt, J.F., Griffin, P.R., Van Duyne, G.D., Spiegelman, B.M.: Disorder-to-order transition underlies the structural basis for the assembly of a transcriptionally active PGC-1α/ERRγ complex. Proc. Natl. Acad. Sci. U. S. A. 108, 18678–18683 (2011)

    Article  CAS  Google Scholar 

  36. Bruning, J.B., Chalmers, M.J., Prasad, S., Busby, S.A., Kamenecka, T.M., He, Y., Nettles, K.W., Griffin, P.R.: Partial agonists activate PPARgamma using a helix 12 independent mechanism. Structure 15, 1258–1271 (2007)

    Article  CAS  Google Scholar 

  37. Jin, L., Martynowski, D., Zheng, S., Wada, T., Xie, W., Li, Y.: Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORγ. Mol. Endocrinol. 24, 923–929 (2010)

    Article  CAS  Google Scholar 

  38. Chalmers, M.J., Busby, S.A., Pascal, B.D., He, Y., Hendrickson, C.L., Marshall, A.G., Griffin, P.R.: Probing protein/ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Anal. Chem. 78, 1005–1014 (2006)

    Article  CAS  Google Scholar 

  39. Busby, S.A., Chalmers, M.J., Griffin, P.R.: Improving digestion efficiency under H/D exchange conditions with activated pepsinogen coupled columns. Int. J. Mass Spectrom. 259, 130–139 (2007)

    Article  CAS  Google Scholar 

  40. Pascal, B.D., Willis, S., Lauer, J.L., Landgraf, R.R., West, G.M., Marciano, D., Novick, S., Goswami, D., Chalmers, M.J., Griffin, P.R.: HDX workbench: software for the analysis of H/D exchange MS data. J. Am. Soc. Mass Spectrom. 23, 1512–1521 (2012)

    Article  CAS  Google Scholar 

  41. West, G.M., Pascal, B.D., Ng, L.M., Soon, F.F., Melcher, K., Xu, H.E., Chalmers, M.J., Griffin, P.R.: Protein conformation ensembles monitored by HDX reveal a structural rationale for abscisic acid signaling protein affinities and activities. Structure 21, 229–235 (2013)

    Article  CAS  Google Scholar 

  42. Puigserver, P., Wu, Z., Park, C.W., Graves, R., Wright, M., Spiegelman, B.M.: A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92, 829–839 (1998)

    Article  CAS  Google Scholar 

  43. Greschik, H., Althage, M., Flaig, R., Sato, Y., Chavant, V., Peluso-Iltis, C., Choulier, L., Cronet, P., Rochel, N., Schule, R., Strömstedt, P.E., Moras, D.: Communication between the ERRα homodimer interface and the PGC-1α binding surface via the helix 8–9 loop. J. Biol. Chem. 283, 20220–20230 (2008)

    Google Scholar 

  44. Li, Y., Kovach, A., Suino-Powell, K., Martynowski, D., Xu, H.E.: Structural and biochemical basis for the binding selectivity of peroxisome proliferator-activated receptor gamma to PGC-1α. J. Biol. Chem. 283, 19132–19139 (2008)

    Article  CAS  Google Scholar 

  45. Englander, S.W., Kallenbach, N.R.: Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q. Rev. Biophys. 16, 521–655 (1983)

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful for support from Ruben Garcia-Ordonez for the protein preparation. The work was supported in part by a Pfizer postdoctoral fellowship and NIGMS (GM084041, to P.R.G.).

Author information

Authors and Affiliations

  1. Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, 33458, USA

    Devrishi Goswami, Michael J. Chalmers, Bruce D. Pascal & Patrick R. Griffin

  2. Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, MA, 02215, USA

    Srikripa Devarakonda & Bruce M. Spiegelman

Authors
  1. Devrishi Goswami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Srikripa Devarakonda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael J. Chalmers
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bruce D. Pascal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bruce M. Spiegelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patrick R. Griffin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Patrick R. Griffin.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Figure 1

Deuterium buildup curves for selected regions of PGC-1α 220. The differential HDX experiment was carried out with PGC-1α 220 in the presence and absence of PPARγ LBD-Rosiglitazone at pH 6.0 (PPTX 97 kb)

Supplementary Figure 2

Deuterium uptake in selected segments of PGC-1α 220 at pH 6.0 and pH 7.5. All time points are converted to pH 7.5 using Equation 1 (also see Table 1). All buildup curves show overlap within the experimental errors indicating the unchanged dynamics of apo PGC-1α 220 at pH 6.0 and 7.5 (PPTX 121 kb)

Supplementary Figure 3

Measurement of thermodynamic stability (Tm) and hence the affinity between PGC-1α 220 and PPARγ. (A) and (B) Representative thermal denaturation curves using Far-UV CD spectra at pH 7.5 and pH 6.0. (C) Tabulated triplicate Tm values, average values and the standard deviation of the binary complex at pH 7.5 and pH 6.0. The differences are not statistically significant. Thermodynamic measurement using CD is performed according to [35] except the wavelength used is 230 nm (PPTX 3287 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Goswami, D., Devarakonda, S., Chalmers, M.J. et al. Time Window Expansion for HDX Analysis of an Intrinsically Disordered Protein. J. Am. Soc. Mass Spectrom. 24, 1584–1592 (2013). https://doi.org/10.1007/s13361-013-0669-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13361-013-0669-y

Key words

Search

Navigation