Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Mar;74(3):1115–1134. doi: 10.1016/S0006-3495(98)77830-0

Loss of conformational stability in calmodulin upon methionine oxidation.

J Gao 1, D H Yin 1, Y Yao 1, H Sun 1, Z Qin 1, C Schöneich 1, T D Williams 1, T C Squier 1
PMCID: PMC1299464  PMID: 9512014

Abstract

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM.

Full Text

The Full Text of this article is available as a PDF (326.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexianu M. E., Ho B. K., Mohamed A. H., La Bella V., Smith R. G., Appel S. H. The role of calcium-binding proteins in selective motoneuron vulnerability in amyotrophic lateral sclerosis. Ann Neurol. 1994 Dec;36(6):846–858. doi: 10.1002/ana.410360608. [DOI] [PubMed] [Google Scholar]
  2. Babu Y. S., Bugg C. E., Cook W. J. Structure of calmodulin refined at 2.2 A resolution. J Mol Biol. 1988 Nov 5;204(1):191–204. doi: 10.1016/0022-2836(88)90608-0. [DOI] [PubMed] [Google Scholar]
  3. Barbato G., Ikura M., Kay L. E., Pastor R. W., Bax A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry. 1992 Jun 16;31(23):5269–5278. doi: 10.1021/bi00138a005. [DOI] [PubMed] [Google Scholar]
  4. Bayley P. M., Martin S. R. The alpha-helical content of calmodulin is increased by solution conditions favouring protein crystallisation. Biochim Biophys Acta. 1992 Nov 10;1160(1):16–21. doi: 10.1016/0167-4838(92)90034-b. [DOI] [PubMed] [Google Scholar]
  5. Becktel W. J., Schellman J. A. Protein stability curves. Biopolymers. 1987 Nov;26(11):1859–1877. doi: 10.1002/bip.360261104. [DOI] [PubMed] [Google Scholar]
  6. Breton C., Chaboud A., Matthys-Rochon E., Bates E. E., Cock J. M., Fromm H., Dumas C. PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis. Plant Mol Biol. 1995 Jan;27(1):105–113. doi: 10.1007/BF00019182. [DOI] [PubMed] [Google Scholar]
  7. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem. 1987;56:395–433. doi: 10.1146/annurev.bi.56.070187.002143. [DOI] [PubMed] [Google Scholar]
  8. Chattopadhyaya R., Meador W. E., Means A. R., Quiocho F. A. Calmodulin structure refined at 1.7 A resolution. J Mol Biol. 1992 Dec 20;228(4):1177–1192. doi: 10.1016/0022-2836(92)90324-d. [DOI] [PubMed] [Google Scholar]
  9. Chin D., Means A. R. Methionine to glutamine substitutions in the C-terminal domain of calmodulin impair the activation of three protein kinases. J Biol Chem. 1996 Nov 29;271(48):30465–30471. doi: 10.1074/jbc.271.48.30465. [DOI] [PubMed] [Google Scholar]
  10. Chye M. L., Liu C. M., Tan C. T. A cDNA clone encoding Brassica calmodulin. Plant Mol Biol. 1995 Jan;27(2):419–423. doi: 10.1007/BF00020195. [DOI] [PubMed] [Google Scholar]
  11. Coyle J. T., Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science. 1993 Oct 29;262(5134):689–695. doi: 10.1126/science.7901908. [DOI] [PubMed] [Google Scholar]
  12. Craig T. A., Watterson D. M., Prendergast F. G., Haiech J., Roberts D. M. Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin. J Biol Chem. 1987 Mar 5;262(7):3278–3284. [PubMed] [Google Scholar]
  13. Crivici A., Ikura M. Molecular and structural basis of target recognition by calmodulin. Annu Rev Biophys Biomol Struct. 1995;24:85–116. doi: 10.1146/annurev.bb.24.060195.000505. [DOI] [PubMed] [Google Scholar]
  14. Dale R. E., Eisinger J., Blumberg W. E. The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer. Biophys J. 1979 May;26(2):161–193. doi: 10.1016/S0006-3495(79)85243-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dale R. E., Eisinger J. Intramolecular energy transfer and molecular conformation. Proc Natl Acad Sci U S A. 1976 Feb;73(2):271–273. doi: 10.1073/pnas.73.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ehrhardt M. R., Erijman L., Weber G., Wand A. J. Molecular recognition by calmodulin: pressure-induced reorganization of a novel calmodulin-peptide complex. Biochemistry. 1996 Feb 6;35(5):1599–1605. doi: 10.1021/bi951267r. [DOI] [PubMed] [Google Scholar]
  17. Fairclough R. H., Cantor C. R. The use of singlet-singlet energy transfer to study macromolecular assemblies. Methods Enzymol. 1978;48:347–379. doi: 10.1016/s0076-6879(78)48019-x. [DOI] [PubMed] [Google Scholar]
  18. Finn B. E., Evenäs J., Drakenberg T., Waltho J. P., Thulin E., Forsén S. Calcium-induced structural changes and domain autonomy in calmodulin. Nat Struct Biol. 1995 Sep;2(9):777–783. doi: 10.1038/nsb0995-777. [DOI] [PubMed] [Google Scholar]
  19. Galaud J. P., Lareyre J. J., Boyer N. Isolation, sequencing and analysis of the expression of Bryonia calmodulin after mechanical perturbation. Plant Mol Biol. 1993 Nov;23(4):839–846. doi: 10.1007/BF00021538. [DOI] [PubMed] [Google Scholar]
  20. Griess E. A., Igloi G. L., Feix G. Isolation and sequence comparison of a maize calmodulin cDNA. Plant Physiol. 1994 Apr;104(4):1467–1468. doi: 10.1104/pp.104.4.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Haas E., Katchalski-Katzir E., Steinberg I. Z. Effect of the orientation of donor and acceptor on the probability of energy transfer involving electronic transitions of mixed polarization. Biochemistry. 1978 Nov 14;17(23):5064–5070. doi: 10.1021/bi00616a032. [DOI] [PubMed] [Google Scholar]
  22. Heidorn D. B., Trewhella J. Comparison of the crystal and solution structures of calmodulin and troponin C. Biochemistry. 1988 Feb 9;27(3):909–915. doi: 10.1021/bi00403a011. [DOI] [PubMed] [Google Scholar]
  23. Hühmer A. F., Gerber N. C., de Montellano P. R., Schöneich C. Peroxynitrite reduction of calmodulin stimulation of neuronal nitric oxide synthase. Chem Res Toxicol. 1996 Mar;9(2):484–491. doi: 10.1021/tx950152l. [DOI] [PubMed] [Google Scholar]
  24. James P., Vorherr T., Carafoli E. Calmodulin-binding domains: just two faced or multi-faceted? Trends Biochem Sci. 1995 Jan;20(1):38–42. doi: 10.1016/s0968-0004(00)88949-5. [DOI] [PubMed] [Google Scholar]
  25. Jena P. K., Reddy A. S., Poovaiah B. W. Molecular cloning and sequencing of a cDNA for plant calmodulin: signal-induced changes in the expression of calmodulin. Proc Natl Acad Sci U S A. 1989 May;86(10):3644–3648. doi: 10.1073/pnas.86.10.3644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johnson J. D. Allosteric interactions among drug binding sites on calmodulin. Biochem Biophys Res Commun. 1983 Apr 29;112(2):787–793. doi: 10.1016/0006-291x(83)91530-9. [DOI] [PubMed] [Google Scholar]
  27. Johnson M. L., Faunt L. M. Parameter estimation by least-squares methods. Methods Enzymol. 1992;210:1–37. doi: 10.1016/0076-6879(92)10003-v. [DOI] [PubMed] [Google Scholar]
  28. Kataoka M., Head J. F., Persechini A., Kretsinger R. H., Engelman D. M. Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation. Biochemistry. 1991 Feb 5;30(5):1188–1192. doi: 10.1021/bi00219a004. [DOI] [PubMed] [Google Scholar]
  29. Khachaturian Z. S. Calcium hypothesis of Alzheimer's disease and brain aging. Ann N Y Acad Sci. 1994 Dec 15;747:1–11. doi: 10.1111/j.1749-6632.1994.tb44398.x. [DOI] [PubMed] [Google Scholar]
  30. Kilhoffer M. C., Kubina M., Travers F., Haiech J. Use of engineered proteins with internal tryptophan reporter groups and pertubation techniques to probe the mechanism of ligand-protein interactions: investigation of the mechanism of calcium binding to calmodulin. Biochemistry. 1992 Sep 1;31(34):8098–8106. doi: 10.1021/bi00149a046. [DOI] [PubMed] [Google Scholar]
  31. Klee C. B. Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase. Biochemistry. 1977 Mar 8;16(5):1017–1024. doi: 10.1021/bi00624a033. [DOI] [PubMed] [Google Scholar]
  32. Kung C., Preston R. R., Maley M. E., Ling K. Y., Kanabrocki J. A., Seavey B. R., Saimi Y. In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli. Cell Calcium. 1992 Jun-Jul;13(6-7):413–425. doi: 10.1016/0143-4160(92)90054-v. [DOI] [PubMed] [Google Scholar]
  33. Lakowicz J. R., Cherek H., Maliwal B. P. Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry. Biochemistry. 1985 Jan 15;24(2):376–383. doi: 10.1021/bi00323a021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ling V., Zielinski R. E. Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.). Plant Physiol. 1989 Jun;90(2):714–719. doi: 10.1104/pp.90.2.714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Luedtke R., Owen C. S., Vanderkooi J. M., Karush F. Proximity relationships within the Fc segment of rabbit immunoglobulin G analyzed by resonance energy transfer. Biochemistry. 1981 May 12;20(10):2927–2936. doi: 10.1021/bi00513a033. [DOI] [PubMed] [Google Scholar]
  36. Lukas T. J., Iverson D. B., Schleicher M., Watterson D. M. Structural characterization of a higher plant calmodulin : spinacia oleracea. Plant Physiol. 1984 Jul;75(3):788–795. doi: 10.1104/pp.75.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mackall J., Klee C. B. Calcium-induced sensitization of the central helix of calmodulin to proteolysis. Biochemistry. 1991 Jul 23;30(29):7242–7247. doi: 10.1021/bi00243a028. [DOI] [PubMed] [Google Scholar]
  38. Maune J. F., Klee C. B., Beckingham K. Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin. J Biol Chem. 1992 Mar 15;267(8):5286–5295. [PubMed] [Google Scholar]
  39. Meador W. E., Means A. R., Quiocho F. A. Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science. 1993 Dec 10;262(5140):1718–1721. doi: 10.1126/science.8259515. [DOI] [PubMed] [Google Scholar]
  40. Meador W. E., Means A. R., Quiocho F. A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. 1992 Aug 28;257(5074):1251–1255. doi: 10.1126/science.1519061. [DOI] [PubMed] [Google Scholar]
  41. Michaelis M. L., Bigelow D. J., Schöneich C., Williams T. D., Ramonda L., Yin D., Hühmer A. F., Yao Y., Gao J., Squier T. C. Decreased plasma membrane calcium transport activity in aging brain. Life Sci. 1996;59(5-6):405–412. doi: 10.1016/0024-3205(96)00319-0. [DOI] [PubMed] [Google Scholar]
  42. Mukherjea P., Maune J. F., Beckingham K. Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin. Protein Sci. 1996 Mar;5(3):468–477. doi: 10.1002/pro.5560050308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nelson D. P., Kiesow L. A. Enthalpy of decomposition of hydrogen peroxide by catalase at 25 degrees C (with molar extinction coefficients of H 2 O 2 solutions in the UV). Anal Biochem. 1972 Oct;49(2):474–478. doi: 10.1016/0003-2697(72)90451-4. [DOI] [PubMed] [Google Scholar]
  44. Pedigo S., Shea M. A. Discontinuous equilibrium titrations of cooperative calcium binding to calmodulin monitored by 1-D 1H-nuclear magnetic resonance spectroscopy. Biochemistry. 1995 Aug 22;34(33):10676–10689. doi: 10.1021/bi00033a044. [DOI] [PubMed] [Google Scholar]
  45. Pedigo S., Shea M. A. Quantitative endoproteinase GluC footprinting of cooperative Ca2+ binding to calmodulin: proteolytic susceptibility of E31 and E87 indicates interdomain interactions. Biochemistry. 1995 Jan 31;34(4):1179–1196. doi: 10.1021/bi00004a011. [DOI] [PubMed] [Google Scholar]
  46. Rosen D. R., Siddique T., Patterson D., Figlewicz D. A., Sapp P., Hentati A., Donaldson D., Goto J., O'Regan J. P., Deng H. X. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993 Mar 4;362(6415):59–62. doi: 10.1038/362059a0. [DOI] [PubMed] [Google Scholar]
  47. Royer C. A. Understanding fluorescence decay in proteins. Biophys J. 1993 Jul;65(1):9–10. doi: 10.1016/S0006-3495(93)81024-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sacks D. B., Lopez M. M., Li Z., Kosk-Kosicka D. Analysis of phosphorylation and mutation of tyrosine residues of calmodulin on its activation of the erythrocyte Ca(2+)-transporting ATPase. Eur J Biochem. 1996 Jul 1;239(1):98–104. doi: 10.1111/j.1432-1033.1996.0098u.x. [DOI] [PubMed] [Google Scholar]
  49. Saxena V. P., Wetlaufer D. B. A new basis for interpreting the circular dichroic spectra of proteins. Proc Natl Acad Sci U S A. 1971 May;68(5):969–972. doi: 10.1073/pnas.68.5.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sayle R. A., Milner-White E. J. RASMOL: biomolecular graphics for all. Trends Biochem Sci. 1995 Sep;20(9):374–374. doi: 10.1016/s0968-0004(00)89080-5. [DOI] [PubMed] [Google Scholar]
  51. Seamon K. B. Calcium- and magnesium-dependent conformational states of calmodulin as determined by nuclear magnetic resonance. Biochemistry. 1980 Jan 8;19(1):207–215. doi: 10.1021/bi00542a031. [DOI] [PubMed] [Google Scholar]
  52. Selkoe D. J. Alzheimer's disease: genotypes, phenotypes, and treatments. Science. 1997 Jan 31;275(5300):630–631. doi: 10.1126/science.275.5300.630. [DOI] [PubMed] [Google Scholar]
  53. Shea M. A., Verhoeven A. S., Pedigo S. Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106. Biochemistry. 1996 Mar 5;35(9):2943–2957. doi: 10.1021/bi951934g. [DOI] [PubMed] [Google Scholar]
  54. Small E. W., Anderson S. R. Fluorescence anisotropy decay demonstrates calcium-dependent shape changes in photo-cross-linked calmodulin. Biochemistry. 1988 Jan 12;27(1):419–428. doi: 10.1021/bi00401a063. [DOI] [PubMed] [Google Scholar]
  55. Smith M. A., Perry G., Richey P. L., Sayre L. M., Anderson V. E., Beal M. F., Kowall N. Oxidative damage in Alzheimer's. Nature. 1996 Jul 11;382(6587):120–121. doi: 10.1038/382120b0. [DOI] [PubMed] [Google Scholar]
  56. Sohal R. S., Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996 Jul 5;273(5271):59–63. doi: 10.1126/science.273.5271.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sorensen B. R., Shea M. A. Calcium binding decreases the stokes radius of calmodulin and mutants R74A, R90A, and R90G. Biophys J. 1996 Dec;71(6):3407–3420. doi: 10.1016/S0006-3495(96)79535-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Starovasnik M. A., Su D. R., Beckingham K., Klevit R. E. A series of point mutations reveal interactions between the calcium-binding sites of calmodulin. Protein Sci. 1992 Feb;1(2):245–253. doi: 10.1002/pro.5560010206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Strasburg G. M., Hogan M., Birmachu W., Thomas D. D., Louis C. F. Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum. J Biol Chem. 1988 Jan 5;263(1):542–548. [PubMed] [Google Scholar]
  60. Takezawa D., Liu Z. H., An G., Poovaiah B. W. Calmodulin gene family in potato: developmental and touch-induced expression of the mRNA encoding a novel isoform. Plant Mol Biol. 1995 Feb;27(4):693–703. doi: 10.1007/BF00020223. [DOI] [PubMed] [Google Scholar]
  61. Thulin E., Andersson A., Drakenberg T., Forsén S., Vogel H. J. Metal ion and drug binding to proteolytic fragments of calmodulin: proteolytic, cadmium-113, and proton nuclear magnetic resonance studies. Biochemistry. 1984 Apr 10;23(8):1862–1870. doi: 10.1021/bi00303a043. [DOI] [PubMed] [Google Scholar]
  62. Toda H., Yazawa M., Sakiyama F., Yagi K. Amino acid sequence of calmodulin from wheat germ. J Biochem. 1994 Feb;115(2):367–367. doi: 10.1093/oxfordjournals.jbchem.a124344. [DOI] [PubMed] [Google Scholar]
  63. Toda H., Yazawa M., Sakiyama F., Yagi K. Amino acid sequence of calmodulin from wheat germ. J Biochem. 1985 Sep;98(3):833–842. doi: 10.1093/oxfordjournals.jbchem.a135342. [DOI] [PubMed] [Google Scholar]
  64. Török K., Lane A. N., Martin S. R., Janot J. M., Bayley P. M. Effects of calcium binding on the internal dynamic properties of bovine brain calmodulin, studied by NMR and optical spectroscopy. Biochemistry. 1992 Apr 7;31(13):3452–3462. doi: 10.1021/bi00128a020. [DOI] [PubMed] [Google Scholar]
  65. Wang C. L., Leavis P. C., Gergely J. Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin. Biochemistry. 1984 Dec 18;23(26):6410–6415. doi: 10.1021/bi00321a020. [DOI] [PubMed] [Google Scholar]
  66. Yang T., Segal G., Abbo S., Feldman M., Fromm H. Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects. Mol Gen Genet. 1996 Oct 28;252(6):684–694. doi: 10.1007/BF02173974. [DOI] [PubMed] [Google Scholar]
  67. Yao Y., Schöneich C., Squier T. C. Resolution of structural changes associated with calcium activation of calmodulin using frequency domain fluorescence spectroscopy. Biochemistry. 1994 Jun 28;33(25):7797–7810. doi: 10.1021/bi00191a007. [DOI] [PubMed] [Google Scholar]
  68. Yao Y., Squier T. C. Variable conformation and dynamics of calmodulin complexed with peptides derived from the autoinhibitory domains of target proteins. Biochemistry. 1996 May 28;35(21):6815–6827. doi: 10.1021/bi960229k. [DOI] [PubMed] [Google Scholar]
  69. Yao Y., Yin D., Jas G. S., Kuczer K., Williams T. D., Schöneich C., Squier T. C. Oxidative modification of a carboxyl-terminal vicinal methionine in calmodulin by hydrogen peroxide inhibits calmodulin-dependent activation of the plasma membrane Ca-ATPase. Biochemistry. 1996 Feb 27;35(8):2767–2787. doi: 10.1021/bi951712i. [DOI] [PubMed] [Google Scholar]
  70. Yoshida M., Minowa O., Yagi K. Divalent cation binding to wheat germ calmodulin. J Biochem. 1983 Dec;94(6):1925–1933. doi: 10.1093/oxfordjournals.jbchem.a134546. [DOI] [PubMed] [Google Scholar]
  71. Zhang M., Li M., Wang J. H., Vogel H. J. The effect of Met-->Leu mutations on calmodulin's ability to activate cyclic nucleotide phosphodiesterase. J Biol Chem. 1994 Jun 3;269(22):15546–15552. [PubMed] [Google Scholar]
  72. Zhang M., Tanaka T., Ikura M. Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nat Struct Biol. 1995 Sep;2(9):758–767. doi: 10.1038/nsb0995-758. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES