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Small Angle X-Ray Scattering Spectroscopy

  • David W. Mulder
  • John W. PetersEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 766)

Abstract

Conformational changes imposed upon the Fe protein during binding and hydrolysis of Mg·ATP are key to initiating the cycle of interactions within the nitrogenase complex that result in gated electron transfer and the eventual multiple electron reduction of dinitrogen to ammonia. Wonderful insights into how nitrogenase accomplishes this have been gleaned from a number of very nice crystal structures, but conformational changes can only be inferred in a very superficial manner, and a number of key conformations relevant to fully understanding conformational changes have eluded this approach. Alternatively, small angle x-ray scattering (SAXS) has proven to be a helpful method for complimenting x-ray crystallography and determining solution structures of various nucleotide-bound states.

Key words

Nitrogenase complex structure conformation SAXS 

Notes

Acknowledgments

Portions of the research carried out used to develop the protocols described in this chapter were conducted out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.

References

  1. 1.
    Bulen WA, LeComte JR (1966) The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci USA 56:979–986PubMedCrossRefGoogle Scholar
  2. 2.
    Hageman RV, Burris RH (1978) Nitrogenase and nitrogenase reductase associate and dissociate with each catalytic cycle. Proc Natl Acad Sci USA 75:2699–2702PubMedCrossRefGoogle Scholar
  3. 3.
    Lanzilotta WN, Parker VD, Seefeldt LC (1998) Electron transfer in nitrogenase analyzed by Marcus theory: evidence for gating by MgATP. Biochemistry 37:399–407PubMedCrossRefGoogle Scholar
  4. 4.
    Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96:2983–3012PubMedCrossRefGoogle Scholar
  5. 5.
    Howard JB, Rees DC (1994) Nitrogenase: a nucleotide-dependent molecular switch. Annu Rev Biochem 63:235–264PubMedCrossRefGoogle Scholar
  6. 6.
    Seefeldt LC, Dean DR (1997) Role of nucleotides in nitrogenase catalysis. Acc Chem Res 30:260–266CrossRefGoogle Scholar
  7. 7.
    Peters JW, Szilagyi RK (2006) Exploring new frontiers of nitrogenase structure and mechanism. Curr Opin Chem Biol 10:101–108PubMedCrossRefGoogle Scholar
  8. 8.
    Lowe DJ, Thorneley RNF (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution. Biochem J 224:895–901PubMedGoogle Scholar
  9. 9.
    Thorneley RNF, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation. Biochem J 224:887–894PubMedGoogle Scholar
  10. 10.
    Barney BM, Laryukhin M, Igarashi RY et al (2005) Trapping a hydrazine reduction intermediate on the nitrogenase active site. Biochemistry 44:8030–8037PubMedCrossRefGoogle Scholar
  11. 11.
    Howard JB, Rees DC (2006) How many metals does it take to fix N2? A mechanistic overview of biological nitrogen fixation. Proc Natl Acad Sci USA 103:17088–17093PubMedCrossRefGoogle Scholar
  12. 12.
    Georgiadis MM, Komiya H, Chakrabarti P et al (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257:1653–1659PubMedCrossRefGoogle Scholar
  13. 13.
    Schindelin H, Kisker C, Schlessman JL et al (1997) Structure of ADP x AIF4(-)-stabilized nitrogenase complex and its implications for signal transduction. Nature 387:370–376PubMedCrossRefGoogle Scholar
  14. 14.
    Schlessman JL, Woo D, Joshua-Tor L et al (1998) Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum. J Mol Biol 280:669–685PubMedCrossRefGoogle Scholar
  15. 15.
    Jang SB, Seefeldt LC, Peters JW (2000) Insights into nucleotide signal transduction in nitrogenase: structure of an iron protein with MgADP bound. Biochemistry 39:14745–14752PubMedCrossRefGoogle Scholar
  16. 16.
    Schmid B, Einsle O, Chiu HJ et al (2002) Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure. Biochemistry 41:15557–15565PubMedCrossRefGoogle Scholar
  17. 17.
    Sen S, Igarashi R, Smith A et al (2004) A conformational mimic of the MgATP-bound “on state” of the nitrogenase iron protein. Biochemistry 43:1787–1797PubMedCrossRefGoogle Scholar
  18. 18.
    Tezcan FA, Kaiser JT, Mustafi D et al (2005) Nitrogenase complexes: multiple docking sites for a nucleotide switch protein. Science 309:1377–1380PubMedCrossRefGoogle Scholar
  19. 19.
    Sen S, Krishnakumar A, McClead J et al (2006) Insights into the role of nucleotide-dependent conformational change in nitrogenase catalysis: structural characterization of the nitrogenase Fe protein Leu127 deletion variant with bound MgATP. J Inorg Biochem 100:1041–1052PubMedCrossRefGoogle Scholar
  20. 20.
    Sarma R, Mulder DW, Brecht E et al (2007) Probing the MgATP-bound conformation of the nitrogenase Fe protein by solution small-angle X-ray scattering. Biochemistry 46:14058–14066PubMedCrossRefGoogle Scholar
  21. 21.
    Ryle MJ, Seefeldt LC (1996) Elucidation of a MgATP signal transduction pathway in the nitrogenase iron protein: formation of a conformation resembling the MgATP-bound state by protein engineering. Biochemistry 35:4766–4775PubMedCrossRefGoogle Scholar
  22. 22.
    Smolsky IL, Liu P, Niebuhr M et al (2007) Biological small-angle X-ray scattering facility at the Stanford Synchrotron Radiation Laboratory. J Appl Cryst 40:s453–s458CrossRefGoogle Scholar
  23. 23.
    Svergun D, Barberato C, Koch M (1995) CRYSOL – a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst 28:768–773CrossRefGoogle Scholar
  24. 24.
    Konarev PV, Volkov VV, Sokolova AV et al (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Cryst 36:1277–1282CrossRefGoogle Scholar
  25. 25.
    Svergun D (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Cryst 25:495–503CrossRefGoogle Scholar
  26. 26.
    Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J 76:2879–2886PubMedCrossRefGoogle Scholar
  27. 27.
    Kozin MB, Svergun DI (2001) Automated matching of high- and low-resolution structural models. J Appl Cryst 34:33–41CrossRefGoogle Scholar
  28. 28.
    Volkov VV, Svergun DI (2003) Uniqueness of ab initio shape determination in small-angle scattering. J Appl Cryst 36:860–864CrossRefGoogle Scholar
  29. 29.
    Krigbaum WR, Kugler FR (1970) Molecular conformation of egg-white lysozyme and bovine alpha-lactalbumin in solution. Biochemistry 9:1216–1223PubMedCrossRefGoogle Scholar
  30. 30.
    Vaney MC, Maignan S, Ries-Kautt M et al (1996) High-resolution structure (1.33 A) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission. Acta Crystallogr D Biol Crystallogr 52:505–517PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryAstrobiology Biogeocatalysis Research Center, Montana State UniversityBozemanUSA

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