Electron spin echo decay as a probe of aminoxyl environment in spin-labeled mutants of human carbonic anhydrase II[hair space]

(Note: The full text of this document is currently only available in the PDF Version )

Mikael Lindgren, Gareth R. Eaton, Sandra S. Eaton, Bengt-Harald Jonsson, Per Hammarström, Magdalena Svensson and Uno Carlsson


Abstract

Genetically-engineered human carbonic anhydrase II mutants have been prepared with cysteine introduced at selected locations and spin-labeled with an aminoxyl (formerly known as nitroxide) radical. Two-pulse electron spin echo data have been obtained for samples in 1∶1 water–glycerol employing a Bruker ESP380E spectrometer. Data obtained at 11 and 40 K are fitted to the function Y(τ) = Y(0)· exp[–(2τ/Tm)x]. Tm = 4.4 to 4.1 µs with x > 2 for labels near the surface, but the decay shape changes to Tm = 2 µs, x = 1 for a label buried in a hydrophobic region of the protein. To identify characteristics of the spin label environment that impact Tm and x, 0.1 to 0.5 mM solutions of aminoxyls are examined in a series of glassy solvents. At these spin label concentrations spin echo dephasing is dominated by interaction with solvent protons. For solvents that do not contain methyl groups 1/Tm increases as solvent proton concentration increases. The smallest values of x and of Tm are observed for solvents with the least sterically hindered methyl groups. In samples of spin-labeled engineered proteins the aminoxyl-probe is generally used to explore local motions near room temperature. The data presented here indicate that the shape of the echo decay obtained at low temperature is a sensitive indicator of the proton environment of the spin-label. The combination of lineshape studies at room temperature and spin echo studies at low temperature provide complementary information in spin labeling studies of protein folding and protein–protein interaction.


References

  1. Biological Magnetic Resonance, eds. L. J. Berliner and J. Reuben, Plenum Press, New York, 1989.
  2. M. Lindgren, M. Svensson, P.-O. Freskgård, U. Carlsson, B.-H. Jonsson, L.-G. Mårtensson and P. Jonasson, J. Chem. Soc., Perkin Trans. 2, 1993, 2003 RSC.
  3. W. L. Hubbell, H. S. Mchaourab, C. Altenbach and M. A. Lietzow, Structure, 1996, 4, 779 CrossRef CAS.
  4. M. Lindgren, M. Svensson, P.-O. Freskgård, U. Carlsson, P. Jonasson, L.-G. Mårtensson and B.-H. Jonsson, Biophys. J., 1995, 69, 202 CAS.
  5. M. Svensson, P. Jonasson, P.-O. Freskgård, B.-H. Jonsson, M. Lindgren, L.-G. Mårtensson, M. Gentile, K. Borén and U. Carlsson, Biochemistry, 1995, 34, 8606 CrossRef CAS.
  6. A. Murray-Brelier and M. E. Goldberg, Proteins: Struct. Funct. Genet., 1989, 6, 395 Search PubMed.
  7. J. H. Freed, in Spin Labeling Theory and Applications, ed. L. J. Berliner, Academic Press, New York, 1976, ch. 3 Search PubMed.
  8. S. S. Eaton and G. R. Eaton, J. Magn. Reson., 1993, A102, 354.
  9. I. M. Brown, in Time Domain Electron Spin Resonance, eds. L. Kevan and R. N. Schwartz, Wiley, 1979, ch. 6 Search PubMed.
  10. K. M. Salikhov and Yu. D. Tsvetkov, in Time Domain Electron Spin Resonance, eds. L. Kevan and R. N. Swartz, Wiley, 1979, ch. 7 Search PubMed.
  11. K. Håkansson, M. Carlsson, L. A. Svensson and A. Liljas, J. Mol. Biol., 1992, 227, 1192 CAS.
  12. J. R. Klauder and P. W. Anderson, Phys. Rev., 1962, 125, 912 CrossRef CAS.
  13. K. Nakagawa, M. B. Candelaria, W. W. C. Chik, S. S. Eaton and G. R. Eaton, J. Magn. Reson., 1992, 98, 81 CAS.
  14. S. A. Dzuba, Yu. D. Tsvetkov and A. G. Maryasov, Chem. Phys. Lett., 1992, 188, 217 CrossRef CAS.
  15. J.-L. Du, G. R. Eaton and S. S. Eaton, J. Magn. Reson., 1996, A119, 240.
  16. A. D. Milov, K. M. Salikhov and Yu. D. Tsvetkov, Sov. Phys. Solid State, 1973, 15, 802 Search PubMed (p. 1187 in Russian).
  17. P. Hu and S. R. Hartmann, J. Magn. Reson., 1974, 15, 226.
  18. W. B. Mims, Phys. Rev., 1974, 9, 1 Search PubMed.
Click here to see how this site uses Cookies. View our privacy policy here.