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.

Global analysis of protein structural changes in complex proteomes

Nature Biotechnology volume 32, pages 1036–1044 (2014)Cite this article

Subjects

Abstract

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ∼300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: LiP-SRM workflow.
Figure 2: LiP-SRM of α-Syn and myoglobin spiked into complex cell extracts.
Figure 3: Global analysis of protein conformational changes.
Figure 4: LiP-SRM analysis of core carbon metabolism upon a shift from glycolytic to gluconeogenic growth.
Figure 5: Structural changes of Cdc19 upon a switch from glucose- to ethanol-based metabolism.
Figure 6: Structural transition of the yeast 14-3-3 protein Bmh1 upon a switch from glucose- to ethanol-based metabolism.

Accession codes

Accessions

Protein Data Bank

References

  1. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    Article  CAS  Google Scholar 

  2. Mischerikow, N. & Heck, A.J. Targeted large-scale analysis of protein acetylation. Proteomics 11, 571–589 (2011).

    Article  CAS  Google Scholar 

  3. Olsen, J.V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

    Article  CAS  Google Scholar 

  4. Goh, C.S., Milburn, D. & Gerstein, M. Conformational changes associated with protein-protein interactions. Curr. Opin. Struct. Biol. 14, 104–109 (2004).

    Article  CAS  Google Scholar 

  5. Wrabl, J.O. et al. The role of protein conformational fluctuations in allostery, function, and evolution. Biophys. Chem. 159, 129–141 (2011).

    Article  CAS  Google Scholar 

  6. Rabiller, M. et al. Proteus in the world of proteins: conformational changes in protein kinases. Arch. Pharm. (Weinheim) 343, 193–206 (2010).

    Article  CAS  Google Scholar 

  7. Fink, A.L. Natively unfolded proteins. Curr. Opin. Struct. Biol. 15, 35–41 (2005).

    Article  CAS  Google Scholar 

  8. Korn, E.D., Carlier, M.F. & Pantaloni, D. Actin polymerization and ATP hydrolysis. Science 238, 638–644 (1987).

    Article  CAS  Google Scholar 

  9. Cox, S., Radzio-Andzelm, E. & Taylor, S.S. Domain movements in protein kinases. Curr. Opin. Struct. Biol. 4, 893–901 (1994).

    Article  CAS  Google Scholar 

  10. Bu, Z. & Callaway, D.J. Proteins move! Protein dynamics and long-range allostery in cell signaling. Adv. Protein Chem. Struct. Biol. 83, 163–221 (2011).

    Article  CAS  Google Scholar 

  11. Chiti, F. & Dobson, C.M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366 (2006).

    Article  CAS  Google Scholar 

  12. Eftink, M.R. Fluorescence techniques for studying protein structure. Methods Biochem. Anal. 35, 127–205 (1991).

    CAS  PubMed  Google Scholar 

  13. Ilari, A. & Savino, C. Protein structure determination by x-ray crystallography. Methods Mol. Biol. 452, 63–87 (2008).

    Article  CAS  Google Scholar 

  14. Pelton, J.T. & McLean, L.R. Spectroscopic methods for analysis of protein secondary structure. Anal. Biochem. 277, 167–176 (2000).

    Article  CAS  Google Scholar 

  15. Heyduk, T. Measuring protein conformational changes by FRET/LRET. Curr. Opin. Biotechnol. 13, 292–296 (2002).

    Article  CAS  Google Scholar 

  16. Sakakibara, D. et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458, 102–105 (2009).

    Article  CAS  Google Scholar 

  17. Herzog, F. et al. Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry. Science 337, 1348–1352 (2012).

    Article  CAS  Google Scholar 

  18. Fontana, A. et al. Probing protein structure by limited proteolysis. Acta Biochim. Pol. 51, 299–321 (2004).

    CAS  PubMed  Google Scholar 

  19. Aceto, A. et al. Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1–1. Int. J. Biochem. Cell Biol. 27, 1033–1041 (1995).

    Article  CAS  Google Scholar 

  20. Dieckmann, R., Pavela-Vrancic, M., von Dohren, H. & Kleinkauf, H. Probing the domain structure and ligand-induced conformational changes by limited proteolysis of tyrocidine synthetase 1. J. Mol. Biol. 288, 129–140 (1999).

    Article  CAS  Google Scholar 

  21. Peng, Z.Y. & Kim, P.S. A protein dissection study of a molten globule. Biochemistry 33, 2136–2141 (1994).

    Article  CAS  Google Scholar 

  22. Polverino de Laureto, P. et al. Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis. J. Mol. Biol. 334, 129–141 (2003).

    Article  CAS  Google Scholar 

  23. Picotti, P., Bodenmiller, B., Mueller, L.N., Domon, B. & Aebersold, R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138, 795–806 (2009).

    Article  CAS  Google Scholar 

  24. Uversky, V.N. Neuropathology, biochemistry, and biophysics of alpha-synuclein aggregation. J. Neurochem. 103, 17–37 (2007).

    CAS  PubMed  Google Scholar 

  25. De Franceschi, G. et al. Molecular insights into the interaction between alpha-synuclein and docosahexaenoic acid. J. Mol. Biol. 394, 94–107 (2009).

    Article  CAS  Google Scholar 

  26. Vilar, M. et al. The fold of alpha-synuclein fibrils. Proc. Natl. Acad. Sci. USA 105, 8637–8642 (2008).

    Article  CAS  Google Scholar 

  27. Kendrew, J.C. et al. A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature 181, 662–666 (1958).

    Article  CAS  Google Scholar 

  28. Eliezer, D. & Wright, P.E. Is apomyoglobin a molten globule? Structural characterization by NMR. J. Mol. Biol. 263, 531–538 (1996).

    Article  CAS  Google Scholar 

  29. Chechik, G. et al. Activity motifs reveal principles of timing in transcriptional control of the yeast metabolic network. Nat. Biotechnol. 26, 1251–1259 (2008).

    Article  CAS  Google Scholar 

  30. DeRisi, J.L., Iyer, V.R. & Brown, P.O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686 (1997).

    Article  CAS  Google Scholar 

  31. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    Article  CAS  Google Scholar 

  32. Zhang, Q.C. et al. Structure-based prediction of protein-protein interactions on a genome-wide scale. Nature 490, 556–560 (2012).

    Article  CAS  Google Scholar 

  33. Apweiler, R. et al. UniProt: the Universal Protein knowledgebase. Nucleic Acids Res. 32, D115–D119 (2004).

    Article  CAS  Google Scholar 

  34. Costenoble, R. et al. Comprehensive quantitative analysis of central carbon and amino-acid metabolism in Saccharomyces cerevisiae under multiple conditions by targeted proteomics. Mol. Syst. Biol. 7, 464 (2011).

    Article  Google Scholar 

  35. Flikweert, M.T. et al. Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12, 247–257 (1996).

    Article  CAS  Google Scholar 

  36. Xu, Y.F. et al. Regulation of yeast pyruvate kinase by ultrasensitive allostery independent of phosphorylation. Mol. Cell 48, 52–62 (2012).

    Article  CAS  Google Scholar 

  37. Kirtley, M.E. & McKay, M. Fructose-1,6-bisphosphate, a regulator of metabolism. Mol. Cell. Biochem. 18, 141–149 (1977).

    Article  CAS  Google Scholar 

  38. Halfmann, R. & Lindquist, S. Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits. Science 330, 629–632 (2010).

    Article  CAS  Google Scholar 

  39. Veisova, D. et al. The C-terminal segment of yeast BMH proteins exhibits different structure compared to other 14–3-3 protein isoforms. Biochemistry 49, 3853–3861 (2010).

    Article  CAS  Google Scholar 

  40. Thakur, S.S. et al. Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell. Proteomics 10, M110 003699 (2011).

    Article  Google Scholar 

  41. Kleifeld, O. et al. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol. 28, 281–288 (2010).

    Article  CAS  Google Scholar 

  42. Amemiya, T., Koike, R., Fuchigami, S., Ikeguchi, M. & Kidera, A. Classification and annotation of the relationship between protein structural change and ligand binding. J. Mol. Biol. 408, 568–584 (2011).

    Article  CAS  Google Scholar 

  43. Hwang, H., Vreven, T., Janin, J. & Weng, Z. Protein-protein docking benchmark version 4.0. Proteins 78, 3111–3114 (2010).

    Article  CAS  Google Scholar 

  44. Zacharias, M. Accounting for conformational changes during protein-protein docking. Curr. Opin. Struct. Biol. 20, 180–186 (2010).

    Article  CAS  Google Scholar 

  45. Narayanaswamy, R. et al. Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation. Proc. Natl. Acad. Sci. USA 106, 10147–10152 (2009).

    Article  CAS  Google Scholar 

  46. Oliveira, A.P. et al. Regulation of yeast central metabolism by enzyme phosphorylation. Mol. Syst. Biol. 8, 623 (2012).

    Article  Google Scholar 

  47. Lomenick, B. et al. Target identification using drug affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. USA 106, 21984–21989 (2009).

    Article  CAS  Google Scholar 

  48. LeVine, H. III. Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 309, 274–284 (1999).

    Article  CAS  Google Scholar 

  49. Antonini, E. & Brunori, M. Hemoglobin and Myoglobin in their Reactions with Ligands (North-Holland Pub. Co., Amsterdam, 1971).

  50. Teale, F.W. Cleavage of the haem-protein link by acid methylethylketone. Biochim. Biophys. Acta 35, 543 (1959).

    Article  CAS  Google Scholar 

  51. Leibundgut, M., Jenni, S., Frick, C. & Ban, N. Structural basis for substrate delivery by acyl carrier protein in the yeast fatty acid synthase. Science 316, 288–290 (2007).

    Article  CAS  Google Scholar 

  52. Lozza, J. Crystallographic and Biochemical Studies of Fungal Fatty Acid Synthase in Complex with Flavonol Inhibitors. PhD thesis, ETH Zurich (2010).

  53. Sherman, F. Getting started with yeast. Methods Enzymol. 350, 3–41 (2002).

    Article  CAS  Google Scholar 

  54. Elias, J.E. & Gygi, S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    Article  CAS  Google Scholar 

  55. Heintz, D. et al. Phosphoproteome exploration reveals a reformatting of cellular processes in response to low sterol biosynthetic capacity in Arabidopsis. J. Proteome Res. 11, 1228–1239 (2012).

    Article  CAS  Google Scholar 

  56. Xie, X. et al. A comparative phosphoproteomic analysis of a human tumor metastasis model using a label-free quantitative approach. Electrophoresis 31, 1842–1852 (2010).

    Article  CAS  Google Scholar 

  57. Chambers, M.C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).

    Article  CAS  Google Scholar 

  58. Glatter, T. et al. Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion. J. Proteome Res. 11, 5145–5156 (2012).

    Article  CAS  Google Scholar 

  59. MacLean, B. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010).

    Article  CAS  Google Scholar 

  60. Choi, M. et al. MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments. Bioinformatics 30, 2524–2526 (2014).

    Article  CAS  Google Scholar 

  61. Storey, J.D. A direct approach to false discovery rates. J. R. Stat. Soc. Series B Stat. Methodol. 64, 479–498 (2002).

    Article  Google Scholar 

  62. Fendt, S.M. et al. Tradeoff between enzyme and metabolite efficiency maintains metabolic homeostasis upon perturbations in enzyme capacity. Mol. Syst. Biol. 6, 356 (2010).

    Article  Google Scholar 

  63. Winston, F., Dollard, C. & Ricupero-Hovasse, S.L. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 53–55 (1995).

    Article  CAS  Google Scholar 

  64. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  Google Scholar 

  65. Berman, H.M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

    Article  CAS  Google Scholar 

  66. Pieper, U. et al. ModBase, a database of annotated comparative protein structure models, and associated resources. Nucleic Acids Res. 39, D465–D474 (2011).

    Article  CAS  Google Scholar 

  67. Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).

    Article  CAS  Google Scholar 

  68. Crooks, G.E., Hon, G., Chandonia, J.M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  Google Scholar 

  69. Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

P.P. is supported by a 'Foerderungsprofessur' grant from the Swiss National Science Foundation (grant PP00P3_133670), by an EU Seventh Framework Program Reintegration grant (FP7-PEOPLE-2010-RG-277147) and by a Promedica Stiftung (grant 2-70669-11). Y.F. is supported by an ETH Research Grant (grant 4412-1); M.S. is supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship D award. G.D.F. is supported by a post-doctoral fellowship of the University of Padua. A.P.O. is supported by the SystemsX.ch project YeastX. We thank R. Costenoble, K. Kochanowski and U. Sauer (ETH Zurich) for insightful discussions and for the measurements of the intracellular concentrations of FBP and M. Peter for access to plasmid and strain collections. We are grateful to O. Vitek and M. Choi (Purdue University), R. Riek, C. Chi and P. Navarro (ETH Zurich) for helpful discussions. We also thank P. Nanni and R. Schlapbach from the Functional Genomics Centre Zurich for access to mass spectrometry instrumentation, F. Allain for access to the D-BIOL Biomolecular NMR Spectroscopy Platform at the ETH Zurich, N. Ban and M.A. Leibundgut for providing a sample of purified yeast fatty acid synthase.

Author information

Author notes

  1. Yuehan Feng, Giorgia De Franceschi and Abdullah Kahraman: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

    Yuehan Feng, Giorgia De Franceschi, Abdullah Kahraman, Martin Soste, Andre Melnik, Paul J Boersema & Paola Picotti

  2. CRIBI Biotechnology Centre, University of Padua, Padua, Italy

    Giorgia De Franceschi & Patrizia Polverino de Laureto

  3. Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland

    Yaroslav Nikolaev

  4. Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

    Ana Paula Oliveira

Authors
  1. Yuehan Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Giorgia De Franceschi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abdullah Kahraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martin Soste
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andre Melnik
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Paul J Boersema
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Patrizia Polverino de Laureto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yaroslav Nikolaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ana Paula Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Paola Picotti
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.P. conceived and supervised the project. Y.F., G.D.F. and P.P. designed and performed the experiments. Y.F., G.D.F., M.S. and A.M. performed experiments and analyzed the data. P.B. contributed to mass spectrometry measurements. P.P.d.L. supervised parts of the project. A.K. analyzed the data. A.P.O. and Y.N. contributed to the analysis and validation of the metabolic data. P.P., Y.F., G.D.F. and A.K. wrote the manuscript.

Corresponding author

Correspondence to Paola Picotti.

Ethics declarations

Competing interests

P.P., Y.F. and G.D.F. are inventors on a patent application that pertains to the method presented in this study.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 2–6 and Supplementary Note (PDF 4522 kb)

Supplementary Table 1

Complete set of SRM assays used in this study (XLSX 514 kb)

Supplementary Table 7

LiP sites identified from spectral count cata (XLSX 664 kb)

Supplementary Table 8

Structural properties of proteins subject to LiP cleavage in the proteome of yeast grown in glucose-based medium (XLSX 628 kb)

Supplementary Table 9

Proteins that change conformational properties upon transition from glucose to ethanol growth conditions (XLSX 81 kb)

Supplementary Table 10

Background and target proteomes used in the functional enrichment analysis (XLSX 64 kb)

Supplementary Table 11

Changes in abundance and in the LiP pattern for metabolic enzymes for yeast grown in ethanol, relative to yeast grown in glucose (XLSX 21 kb)

Supplementary Table 12

Changes in the LiP pattern of TCA cycle enzymes (XLSX 43 kb)

Supplementary Table 13

Changes in the LiP pattern of pyruvate kinase (XLSX 15 kb)

Supplementary Table 14

Reversion of the proteolytic pattern of Cdc19 by addition of FBP to the extract from cells grown in ethanol (XLSX 14 kb)

Supplementary Table 15

Changes in LiP pattern of proteins in the proteome of yeast grown in ethanol upon administration of FBP (XLSX 44 kb)

Supplementary Table 16

Changes in the LiP pattern of fatty acid synthase subunits 1 and 2 (Fas1 and Fas2) upon FBP administration (XLSX 82 kb)

Supplementary Table 17

Changes in the LiP pattern of Bmh1 and Bmh2 (XLSX 13 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Feng, Y., De Franceschi, G., Kahraman, A. et al. Global analysis of protein structural changes in complex proteomes. Nat Biotechnol 32, 1036–1044 (2014). https://doi.org/10.1038/nbt.2999

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2999

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research