Skip to main content
SpringerLink
Log in

Structures of Dynamic Protein Complexes: Hybrid Techniques to Study MAP Kinase Complexes and the ESCRT System

  • Protocol
  • First Online:
Protein NMR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1688))

Abstract

The integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Blanchet CE, Svergun DI (2013) Small-angle X-ray scattering on biological macromolecules and nanocomposites in solution. Annu Rev Phys Chem 64:37–54. doi:10.1146/annurev-physchem-040412-110132

    Article  CAS  PubMed  Google Scholar 

  2. Graewert MA, Svergun DI (2013) Impact and progress in small and wide angle X-ray scattering (SAXS and WAXS). Curr Opin Struct Biol 23(5):748–754. doi:10.1016/j.sbi.2013.06.007

    Article  CAS  PubMed  Google Scholar 

  3. Bernado P, Perez Y, Svergun DI, Pons M (2008) Structural characterization of the active and inactive states of Src kinase in solution by small-angle X-ray scattering. J Mol Biol 376(2):492–505. doi:10.1016/j.jmb.2007.11.066

    Article  CAS  PubMed  Google Scholar 

  4. Pelikan M, Hura GL, Hammel M (2009) Structure and flexibility within proteins as identified through small angle X-ray scattering. Gen Physiol Biophys 28(2):174–189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Alber F, Forster F, Korkin D, Topf M, Sali A (2008) Integrating diverse data for structure determination of macromolecular assemblies. Annu Rev Biochem 77:443–477. doi:10.1146/annurev.biochem.77.060407.135530

    Article  CAS  PubMed  Google Scholar 

  6. Rozycki B, Kim YC, Hummer G (2011) SAXS ensemble refinement of ESCRT-III CHMP3 conformational transitions. Structure 19(1):109–116. doi:10.1016/j.str.2010.10.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Yang S, Blachowicz L, Makowski L, Roux B (2010) Multidomain assembled states of Hck tyrosine kinase in solution. Proc Natl Acad Sci USA 107(36):15757–15762. doi:10.1073/pnas.1004569107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Peti W, Page R (2016) NMR spectroscopy to study MAP kinase binding to MAP kinase phosphatases. Meth Mol Biol 1447:181–196. doi:10.1007/978-1-4939-3746-2_11

    Article  Google Scholar 

  9. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16(1):18–29. doi:10.1038/nrm3920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51(1):1–10. doi:10.1016/j.pep.2006.06.024

    Article  CAS  PubMed  Google Scholar 

  11. Svergun DI, Barberato C, Koch MHJ (1995) CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst 28:768–773

    Google Scholar 

  12. Schneidman-Duhovny D, Hammel M, Sali A (2010) FoXS: a web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res 38(Web Server issue):W540–W544. doi:10.1093/nar/gkq461

  13. Grishaev A, Guo L, Irving T, Bax A (2010) Improved fitting of solution X-ray scattering data to macromolecular structures and structural ensembles by explicit water modeling. J Am Chem Soc 132(44):15484–15486. doi:10.1021/ja106173n

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Poitevin F, Orland H, Doniach S, Koehl P, Delarue M (2011) AquaSAXS: a web server for computation and fitting of SAXS profiles with non-uniformally hydrated atomic models. Nucleic Acids Res 39(Web Server issue):W184–W189. doi:10.1093/nar/gkr430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu HG, Hexemer A, Zwart PH (2012) The small angle scattering ToolBox (SASTBX): an open-source software for biomolecular small-angle scattering. J Appl Cryst 45:587–593

    Article  CAS  Google Scholar 

  16. Kikhney AG, Svergun DI (2015) A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett 589(19 Pt A):2570–2577. doi:10.1016/j.febslet.2015.08.027

    Article  CAS  PubMed  Google Scholar 

  17. Petoukhov MV, Franke D, Shkumatov AV, Tria G, Kikhney AG, Gajda M, Gorba C, Mertens HD, Konarev PV, Svergun DI (2012) New developments in the ATSAS program package for small-angle scattering data analysis. J Appl Cryst 45(Pt 2):342–350. doi:10.1107/S0021889812007662

    Article  CAS  Google Scholar 

  18. Ravikumar KM, Huang W, Yang S (2013) Fast-SAXS-pro: a unified approach to computing SAXS profiles of DNA, RNA, protein, and their complexes. J Chem Phys 138(2):024112. doi:10.1063/1.4774148

    Article  PubMed  Google Scholar 

  19. Rambo RP, Tainer JA (2011) Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95(8):559–571. doi:10.1002/bip.21638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Boura E, Rozycki B, Herrick DZ, Chung HS, Vecer J, Eaton WA, Cafiso DS, Hummer G, Hurley JH (2011) Solution structure of the ESCRT-I complex by small-angle X-ray scattering, EPR, and FRET spectroscopy. Proc Natl Acad Sci USA 108(23):9437–9442. doi:10.1073/pnas.1101763108

  21. Francis DM, Rozycki B, Koveal D, Hummer G, Page R, Peti W (2011) Structural basis of p38alpha regulation by hematopoietic tyrosine phosphatase. Nat Chem Biol 7(12):916–924. doi:10.1038/nchembio.707

    Article  CAS  PubMed  Google Scholar 

  22. Svergun DI, Petoukhov MV, Koch MH (2001) Determination of domain structure of proteins from X-ray solution scattering. Biophys J 80(6):2946–2953. doi:10.1016/S0006-3495(01)76260-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Franke D, Svergun DI (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Cryst 42(Pt 2):342–346. doi:10.1107/S0021889809000338

    Article  CAS  Google Scholar 

  24. Rozycki B, Boura E (2014) Large, dynamic, multi-protein complexes: a challenge for structural biology. J Phys Condens Matter 26(46):463103. doi:10.1088/0953-8984/26/46/463103

    Article  PubMed  Google Scholar 

  25. Boura E, Rozycki B, Chung HS, Herrick DZ, Canagarajah B, Cafiso DS, Eaton WA, Hummer G, Hurley JH (2012) Solution structure of the ESCRT-I and -II supercomplex: implications for membrane budding and scission. Structure 20(5):874–886. doi:10.1016/j.str.2012.03.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Francis DM, Rozycki B, Tortajada A, Hummer G, Peti W, Page R (2011) Resting and active states of the ERK2:HePTP complex. J Am Chem Soc 133(43):17138–17141. doi:10.1021/ja2075136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fiser A, Do RK, Sali A (2000) Modeling of loops in protein structures. Protein Sci 9(9):1753–1773. doi:10.1110/ps.9.9.1753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kim YC, Hummer G (2008) Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding. J Mol Biol 375(5):1416–1433. doi:10.1016/j.jmb.2007.11.063

    Article  CAS  PubMed  Google Scholar 

  29. Kenzaki H, Koga N, Hori N, Kanada R, Li W, Okazaki K, Yao XQ, Takada S (2011) CafeMol: a coarse-grained biomolecular simulator for simulating proteins at work. J Chem Theory Comput 7(6):1979–1989. doi:10.1021/ct2001045

    Article  CAS  PubMed  Google Scholar 

  30. Liwo A, Baranowski M, Czaplewski C, Golas E, He Y, Jagiela D, Krupa P, Maciejczyk M, Makowski M, Mozolewska MA, Niadzvedtski A, Oldziej S, Scheraga HA, Sieradzan AK, Slusarz R, Wirecki T, Yin Y, Zaborowski B (2014) A unified coarse-grained model of biological macromolecules based on mean-field multipole-multipole interactions. J Mol Model 20(8):2306. doi:10.1007/s00894-014-2306-5

    Article  PubMed  PubMed Central  Google Scholar 

  31. Dannenhoffer-Lafage T, White AD, Voth GA (2016) A direct method for incorporating experimental data into multiscale coarse-grained models. J Chem Theory Comput 12(5):2144–2153. doi:10.1021/acs.jctc.6b00043

    Article  CAS  PubMed  Google Scholar 

  32. Yang S, Park S, Makowski L, Roux B (2009) A rapid coarse residue-based computational method for x-ray solution scattering characterization of protein folds and multiple conformational states of large protein complexes. Biophys J 96(11):4449–4463. doi:10.1016/j.bpj.2009.03.036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Polyhach Y, Bordignon E, Jeschke G (2011) Rotamer libraries of spin labelled cysteines for protein studies. Phys Chem Chem Phys 13(6):2356–2366. doi:10.1039/c0cp01865a

    Article  CAS  PubMed  Google Scholar 

  34. Best RB, Merchant KA, Gopich IV, Schuler B, Bax A, Eaton WA (2007) Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Proc Natl Acad Sci USA 104(48):18964–18969. doi:10.1073/pnas.0709567104

  35. Merchant KA, Best RB, Louis JM, Gopich IV, Eaton WA (2007) Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. Proc Natl Acad Sci USA 104(5):1528–1533. doi:10.1073/pnas.0607097104

  36. Hartigan JA, Wong MA (1979) A k-means clustering algorithm. Appl Stat 28:100–108

    Article  Google Scholar 

  37. Heyer LJ, Kruglyak S, Yooseph S (1999) Exploring expression data: identification and analysis of coexpressed genes. Genome Res 9(11):1106–1115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Leung HT, Bignucolo O, Aregger R, Dames SA, Mazur A, Berneche S, Grzesiek S (2016) A rigorous and efficient method to reweight very large conformational ensembles using average experimental data and to determine their relative information content. J Chem Theory Comput 12(1):383–394. doi:10.1021/acs.jctc.5b00759

    Article  CAS  PubMed  Google Scholar 

  39. Rozycki B, Cieplak M, Czjzek M (2015) Large conformational fluctuations of the multi-domain xylanase Z of clostridium thermocellum. J Struct Biol 191(1):68–75. doi:10.1016/j.jsb.2015.05.004

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank all the members of the Page and Peti laboratory. This work was supported by NIH grant R01GM098482 to RP; NIH R01GM100910 and American Diabetes Association Pathway to the Cure 1-14-ACN-31 to WP. EB was supported by the Czech Science Foundation grant number 17-05200S, by the project InterBioMed LO1302 from the Ministry of Education of the Czech Republic and by the Academy of Sciences of the Czech Republic (RVO: 61388963). BR was supported by the National Science Centre, Poland, grant number 2016/21/B/NZ1/00006, and by the European Framework Programme VII NMP grant 604530-2 (CellulosomePlus) and co-financed by the Polish Ministry of Science and Higher Education from the resources granted for the years 2014-2017 in support of scientific projects.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, USA

    Wolfgang Peti & Rebecca Page

  2. Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, 16610, Prague, Czech Republic

    Evzen Boura

  3. Institute of Physics, Polish Academy of Sciences, 02668, Warsaw, Poland

    Bartosz Różycki

Authors
  1. Wolfgang Peti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebecca Page
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evzen Boura
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bartosz Różycki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wolfgang Peti .

Editor information

Editors and Affiliations

  1. Department of Chemistry and Biochemistry, The City College of New York, New York, New York, USA

    Ranajeet Ghose

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Peti, W., Page, R., Boura, E., Różycki, B. (2018). Structures of Dynamic Protein Complexes: Hybrid Techniques to Study MAP Kinase Complexes and the ESCRT System. In: Ghose, R. (eds) Protein NMR. Methods in Molecular Biology, vol 1688. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7386-6_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7386-6_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7385-9

  • Online ISBN: 978-1-4939-7386-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation