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Small-Angle X-Ray Scattering for the Study of Proteins in the Ubiquitin Pathway

  • Jean-François TrempeEmail author
  • Kalle GehringEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)

Abstract

Small-angle X-ray scattering (SAXS) is an invaluable complement to other biophysical methods used to interrogate the structure and dynamics of proteins. Here, we describe the standard experimental protocol used in our laboratory to analyze proteins in the ubiquitin pathway. The method addresses buffer selection, data collection using an in-house X-ray source, diagnostic tests to assess data quality, and computational approaches to interpret SAXS data.

Key words

X-ray scattering SAXS Ubiquitin Kratky plot Guinier analysis Pair-distance distribution function 

References

  1. 1.
    Meisburger SP, Taylor AB, Khan CA, Zhang S, Fitzpatrick PF, Ando N (2016) Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled small-angle X-ray scattering. J Am Chem Soc 138(20):6506–6516.  https://doi.org/10.1021/jacs.6b01563CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Nielsen SS, Toft KN, Snakenborg D, Jeppesen MG, Jacobsen JK, Vestergaard B, Kutter JP, Arleth L (2009) BioXTAS RAW, a software program for high-throughput automated small-angle X-ray scattering data reduction and preliminary analysis. J Appl Crystallogr 42:965–974CrossRefGoogle Scholar
  3. 3.
    Rasool S, Soya N, Truong L, Croteau N, Lukacs GL, Trempe JF (2018) PINK1 autophosphorylation is required for ubiquitin recognition. EMBO reports 19(4):e44981Google Scholar
  4. 4.
    Trempe JF, Sauve V, Grenier K, Seirafi M, Tang MY, Menade M, Al-Abdul-Wahid S, Krett J, Wong K, Kozlov G, Nagar B, Fon EA, Gehring K (2013) Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340:1451–1455. science.1237908 [pii].  https://doi.org/10.1126/science.1237908CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Sauvé V, Lilov A, Seirafi M, Vranas M, Rasool S, Kozlov G, Sprules T, Wang J, Trempe JF, Gehring K (2015) A Ubl/ubiquitin switch in the activation of Parkin. EMBO J 34(20):2492–2505.  https://doi.org/10.15252/embj.201592237CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Taherbhoy AM, Tait SW, Kaiser SE, Williams AH, Deng A, Nourse A, Hammel M, Kurinov I, Rock CO, Green DR, Schulman BA (2011) Atg8 transfer from Atg7 to Atg3: a distinctive E1-E2 architecture and mechanism in the autophagy pathway. Mol Cell 44(3):451–461 S1097-2765(11)00767-2 [pii].  https://doi.org/10.1016/j.molcel.2011.08.034CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Trempe JF, Saskova KG, Siva M, Ratcliffe CD, Veverka V, Hoegl A, Menade M, Feng X, Shenker S, Svoboda M, Kozisek M, Konvalinka J, Gehring K (2016) Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family. Sci Rep 6:33671.  https://doi.org/10.1038/srep33671CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Siva M, Svoboda M, Veverka V, Trempe JF, Hofmann K, Kozisek M, Hexnerova R, Sedlak F, Belza J, Brynda J, Sacha P, Hubalek M, Starkova J, Flaisigova I, Konvalinka J, Saskova KG (2016) Human DNA-damage-inducible 2 protein is structurally and functionally distinct from its yeast Ortholog. Sci Rep 6:30443.  https://doi.org/10.1038/srep30443CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pruneda JN, Stoll KE, Bolton LJ, Brzovic PS, Klevit RE (2011) Ubiquitin in motion: structural studies of the ubiquitin-conjugating enzyme approximately ubiquitin conjugate. Biochemistry 50(10):1624–1633.  https://doi.org/10.1021/bi101913mCrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Davis-Searles PR, Saunders AJ, Erie DA, Winzor DJ, Pielak GJ (2001) Interpreting the effects of small uncharged solutes on protein-folding equilibria. Annu Rev Biophys Biomol Struct 30:271–306.  https://doi.org/10.1146/annurev.biophys.30.1.271CrossRefPubMedGoogle Scholar
  11. 11.
    Grishaev A (2012) Sample preparation, data collection, and preliminary data analysis in biomolecular solution X-ray scattering. Curr Protoc Protein Sci Chapter 17:Unit17.14.  https://doi.org/10.1002/0471140864.ps1714s70CrossRefPubMedGoogle Scholar
  12. 12.
    Petoukhov MV, Franke D, Shkumatov AV, Tria G, Kikhney AG, Gajda M, Gorba C, Haydyn MDT, Konarev PV, Svergun DI (2012) New developments in the ATSAS program package for small-angle scattering data analysis. J Appl Crystallogr 45:342–350CrossRefGoogle Scholar
  13. 13.
    Jacques DA, Trewhella J (2010) Small-angle scattering for structural biology--expanding the frontier while avoiding the pitfalls. Protein Sci 19(4):642–657.  https://doi.org/10.1002/pro.351CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Rambo RP, Tainer JA (2013) Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496(7446):477–481.  https://doi.org/10.1038/nature12070CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Semisotnov GV, Kihara H, Kotova NV, Kimura K, Amemiya Y, Wakabayashi K, Serdyuk IN, Timchenko AA, Chiba K, Nikaido K, Ikura T, Kuwajima K (1996) Protein globularization during folding. A study by synchrotron small-angle X-ray scattering. J Mol Biol 262(4):559–574.  https://doi.org/10.1006/jmbi.1996.0535CrossRefPubMedGoogle Scholar
  16. 16.
    Tsutakawa SE, Hura GL, Frankel KA, Cooper PK, Tainer JA (2007) Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography. J Struct Biol 158(2):214–223.  https://doi.org/10.1016/j.jsb.2006.09.008CrossRefPubMedGoogle Scholar
  17. 17.
    Bernado P, Mylonas E, Petoukhov MV, Blackledge M, Svergun DI (2007) Structural characterization of flexible proteins using small-angle X-ray scattering. J Am Chem Soc 129(17):5656–5664.  https://doi.org/10.1021/ja069124nCrossRefPubMedGoogle Scholar
  18. 18.
    Wriggers W, Chacon P (2001) Using Situs for the registration of protein structures with low-resolution bead models from X-ray solution scattering. J Appl Crystallogr 34:773–776CrossRefGoogle Scholar
  19. 19.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera – a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612.  https://doi.org/10.1002/jcc.20084CrossRefGoogle Scholar
  20. 20.
    Trempe JF, Shenker S, Kozlov G, Gehring K (2011) Self-association studies of the bifunctional N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli. Protein Sci 20(4):745–752.  https://doi.org/10.1002/pro.608CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Svergun DI, Barberato C, Koch MHJ (1995) CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmacology and TherapeuticsMcGill UniversityMontréalCanada
  2. 2.Department of BiochemistryMcGill UniversityMontréalCanada

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