Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Jan;8(1):113–121. doi: 10.1110/ps.8.1.113

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: biochemical and spectroscopic studies.

T Yuan 1, H J Vogel 1
PMCID: PMC2144098  PMID: 10210190

Abstract

Calmodulin (CaM) is a 148-residue regulatory calcium-binding protein that activates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two "active sites" of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium-dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM-binding domain of myosin light-chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild-type CaM (WT-CaM). One- and two-dimensional NMR studies of the Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth-CaM and Nle-CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the K(D) for Nle-CaM also increased 3.5-fold compared with WT-CaM. These data show that the "active site" Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM.

Full Text

The Full Text of this article is available as a PDF (378.1 KB).

Selected References

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

  1. Alix J. H., Hayes D., Nierhaus K. H. Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine. V. Methylation dependence on the assembly of E. coli 50 S ribosomal subunits. J Mol Biol. 1979 Feb 5;127(4):375–395. doi: 10.1016/0022-2836(79)90228-6. [DOI] [PubMed] [Google Scholar]
  2. Anfinsen C. B., Corley L. G. An active variant of staphylococcal nuclease containing norleucine in place of methionine. J Biol Chem. 1969 Oct 10;244(19):5149–5152. [PubMed] [Google Scholar]
  3. Apostol I., Levine J., Lippincott J., Leach J., Hess E., Glascock C. B., Weickert M. J., Blackmore R. Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli. J Biol Chem. 1997 Nov 14;272(46):28980–28988. doi: 10.1074/jbc.272.46.28980. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Barker D. G., Bruton C. J. The fate of norleucine as a replacement for methionine in protein synthesis. J Mol Biol. 1979 Sep 15;133(2):217–231. doi: 10.1016/0022-2836(79)90531-x. [DOI] [PubMed] [Google Scholar]
  7. Blumenthal D. K., Takio K., Edelman A. M., Charbonneau H., Titani K., Walsh K. A., Krebs E. G. Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Proc Natl Acad Sci U S A. 1985 May;82(10):3187–3191. doi: 10.1073/pnas.82.10.3187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bogosian G., Violand B. N., Dorward-King E. J., Workman W. E., Jung P. E., Kane J. F. Biosynthesis and incorporation into protein of norleucine by Escherichia coli. J Biol Chem. 1989 Jan 5;264(1):531–539. [PubMed] [Google Scholar]
  9. Brown J. L. The modification of the amino terminal region of Escherichia coli proteins after initiation with methionine analogues. Biochim Biophys Acta. 1973 Feb 4;294(1):527–529. doi: 10.1016/0005-2787(73)90108-1. [DOI] [PubMed] [Google Scholar]
  10. Budisa N., Karnbrock W., Steinbacher S., Humm A., Prade L., Neuefeind T., Moroder L., Huber R. Bioincorporation of telluromethionine into proteins: a promising new approach for X-ray structure analysis of proteins. J Mol Biol. 1997 Jul 25;270(4):616–623. doi: 10.1006/jmbi.1997.1132. [DOI] [PubMed] [Google Scholar]
  11. Budisa N., Minks C., Medrano F. J., Lutz J., Huber R., Moroder L. Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V. Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):455–459. doi: 10.1073/pnas.95.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Budisa N., Steipe B., Demange P., Eckerskorn C., Kellermann J., Huber R. High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. Eur J Biochem. 1995 Jun 1;230(2):788–796. doi: 10.1111/j.1432-1033.1995.tb20622.x. [DOI] [PubMed] [Google Scholar]
  13. Burgess W. H. Characterization of calmodulin and calmodulin isotypes from sea urchin gametes. J Biol Chem. 1982 Feb 25;257(4):1800–1804. [PubMed] [Google Scholar]
  14. 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]
  15. Chin D., Sloan D. J., Quiocho F. A., Means A. R. Functional consequences of truncating amino acid side chains located at a calmodulin-peptide interface. J Biol Chem. 1997 Feb 28;272(9):5510–5513. doi: 10.1074/jbc.272.9.5510. [DOI] [PubMed] [Google Scholar]
  16. Chin D., Winkler K. E., Means A. R. Characterization of substrate phosphorylation and use of calmodulin mutants to address implications from the enzyme crystal structure of calmodulin-dependent protein kinase I. J Biol Chem. 1997 Dec 12;272(50):31235–31240. doi: 10.1074/jbc.272.50.31235. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Edwards R. A., Walsh M. P., Sutherland C., Vogel H. J. Activation of calcineurin and smooth muscle myosin light chain kinase by Met-to-Leu mutants of calmodulin. Biochem J. 1998 Apr 1;331(Pt 1):149–152. doi: 10.1042/bj3310149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Falke J. J., Drake S. K., Hazard A. L., Peersen O. B. Molecular tuning of ion binding to calcium signaling proteins. Q Rev Biophys. 1994 Aug;27(3):219–290. doi: 10.1017/s0033583500003012. [DOI] [PubMed] [Google Scholar]
  20. Furter R. Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli. Protein Sci. 1998 Feb;7(2):419–426. doi: 10.1002/pro.5560070223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gellman S. H. On the role of methionine residues in the sequence-independent recognition of nonpolar protein surfaces. Biochemistry. 1991 Jul 9;30(27):6633–6636. doi: 10.1021/bi00241a001. [DOI] [PubMed] [Google Scholar]
  22. Gilles A. M., Marlière P., Rose T., Sarfati R., Longin R., Meier A., Fermandjian S., Monnot M., Cohen G. N., Bârzu O. Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase. J Biol Chem. 1988 Jun 15;263(17):8204–8209. [PubMed] [Google Scholar]
  23. Hiraoki T., Vogel H. J. Structure and function of calcium-binding proteins. J Cardiovasc Pharmacol. 1987;10 (Suppl 1):S14–S31. doi: 10.1097/00005344-198710001-00004. [DOI] [PubMed] [Google Scholar]
  24. Hortin G., Boime I. Applications of amino acid analogs for studying co- and posttranslational modifications of proteins. Methods Enzymol. 1983;96:777–784. doi: 10.1016/s0076-6879(83)96065-2. [DOI] [PubMed] [Google Scholar]
  25. Ikura M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci. 1996 Jan;21(1):14–17. [PubMed] [Google Scholar]
  26. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  27. Ikura M., Spera S., Barbato G., Kay L. E., Krinks M., Bax A. Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy. Biochemistry. 1991 Sep 24;30(38):9216–9228. doi: 10.1021/bi00102a013. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Kerwar S. S., Weissbach H. Studies on the ability of norleucine to replace methionine in the initiation of protein synthesis of E. coli. Arch Biochem Biophys. 1970 Dec;141(2):525–532. doi: 10.1016/0003-9861(70)90170-0. [DOI] [PubMed] [Google Scholar]
  30. Koide H., Yokoyama S., Kawai G., Ha J. M., Oka T., Kawai S., Miyake T., Fuwa T., Miyazawa T. Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2-aminohexanoic acid at position 21 in human epidermal growth factor. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6237–6241. doi: 10.1073/pnas.85.17.6237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kuboniwa H., Tjandra N., Grzesiek S., Ren H., Klee C. B., Bax A. Solution structure of calcium-free calmodulin. Nat Struct Biol. 1995 Sep;2(9):768–776. doi: 10.1038/nsb0995-768. [DOI] [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Matthews B. W. Structural and genetic analysis of the folding and function of T4 lysozyme. FASEB J. 1996 Jan;10(1):35–41. doi: 10.1096/fasebj.10.1.8566545. [DOI] [PubMed] [Google Scholar]
  34. Matthews B. W. Studies on protein stability with T4 lysozyme. Adv Protein Chem. 1995;46:249–278. doi: 10.1016/s0065-3233(08)60337-x. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Müller S., Senn H., Gsell B., Vetter W., Baron C., Böck A. The formation of diselenide bridges in proteins by incorporation of selenocysteine residues: biosynthesis and characterization of (Se)2-thioredoxin. Biochemistry. 1994 Mar 22;33(11):3404–3412. doi: 10.1021/bi00177a034. [DOI] [PubMed] [Google Scholar]
  38. Nelson M. R., Chazin W. J. An interaction-based analysis of calcium-induced conformational changes in Ca2+ sensor proteins. Protein Sci. 1998 Feb;7(2):270–282. doi: 10.1002/pro.5560070206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. O'Neil K. T., DeGrado W. F. How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci. 1990 Feb;15(2):59–64. doi: 10.1016/0968-0004(90)90177-d. [DOI] [PubMed] [Google Scholar]
  40. Old J. M., Jones D. S. A comparison of ethionine with methionine in Escherichia coli in vitro polypeptide chain initiation and synthesis. FEBS Lett. 1976 Jul 15;66(2):264–268. doi: 10.1016/0014-5793(76)80519-4. [DOI] [PubMed] [Google Scholar]
  41. Old J. M., Jones D. S. The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase. Biochem J. 1977 Aug 1;165(2):367–373. doi: 10.1042/bj1650367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Padmanabhan S., Baldwin R. L. Straight-chain non-polar amino acids are good helix-formers in water. J Mol Biol. 1991 May 20;219(2):135–137. doi: 10.1016/0022-2836(91)90553-i. [DOI] [PubMed] [Google Scholar]
  43. Parsons J. F., Xiao G., Gilliland G. L., Armstrong R. N. Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan. Biochemistry. 1998 May 5;37(18):6286–6294. doi: 10.1021/bi980219e. [DOI] [PubMed] [Google Scholar]
  44. RICHMOND M. H. The effect of amino acid analogues on growth and protein synthesis in microorganisms. Bacteriol Rev. 1962 Dec;26:398–420. doi: 10.1128/br.26.4.398-420.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Randhawa Z. I., Witkowska H. E., Cone J., Wilkins J. A., Hughes P., Yamanishi K., Yasuda S., Masui Y., Arthur P., Kletke C. Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-CSF, 4-153) expressed in Escherichia coli: structural analysis. Biochemistry. 1994 Apr 12;33(14):4352–4362. doi: 10.1021/bi00180a032. [DOI] [PubMed] [Google Scholar]
  46. Siivari K., Zhang M., Palmer A. G., 3rd, Vogel H. J. NMR studies of the methionine methyl groups in calmodulin. FEBS Lett. 1995 Jun 12;366(2-3):104–108. doi: 10.1016/0014-5793(95)00504-3. [DOI] [PubMed] [Google Scholar]
  47. Strynadka N. C., James M. N. Crystal structures of the helix-loop-helix calcium-binding proteins. Annu Rev Biochem. 1989;58:951–998. doi: 10.1146/annurev.bi.58.070189.004511. [DOI] [PubMed] [Google Scholar]
  48. Thomson J., Ratnaparkhi G. S., Varadarajan R., Sturtevant J. M., Richards F. M. Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the M13Nle mutation in ribonuclease-S. Biochemistry. 1994 Jul 19;33(28):8587–8593. doi: 10.1021/bi00194a025. [DOI] [PubMed] [Google Scholar]
  49. Trupin J., Dickerman H., Nirenberg M., Weissbach H. Formylation of amino acid analogues of methionine sRNA. Biochem Biophys Res Commun. 1966 Jul 6;24(1):50–55. doi: 10.1016/0006-291x(66)90408-6. [DOI] [PubMed] [Google Scholar]
  50. Vogel H. J. The Merck Frosst Award Lecture 1994. Calmodulin: a versatile calcium mediator protein. Biochem Cell Biol. 1994 Sep-Oct;72(9-10):357–376. [PubMed] [Google Scholar]
  51. Vogel H. J., Zhang M. Protein engineering and NMR studies of calmodulin. Mol Cell Biochem. 1995 Aug-Sep;149-150:3–15. doi: 10.1007/BF01076558. [DOI] [PubMed] [Google Scholar]
  52. Waltersson Y., Linse S., Brodin P., Grundström T. Mutational effects on the cooperativity of Ca2+ binding in calmodulin. Biochemistry. 1993 Aug 10;32(31):7866–7871. doi: 10.1021/bi00082a005. [DOI] [PubMed] [Google Scholar]
  53. Wilson M. J., Hatfield D. L. Incorporation of modified amino acids into proteins in vivo. Biochim Biophys Acta. 1984 Apr 5;781(3):205–215. doi: 10.1016/0167-4781(84)90085-x. [DOI] [PubMed] [Google Scholar]
  54. YOSHIDA A., YAMASAKI M. Studies on the mechanism of protein synthesis; incorporation of ethionine into alpha-amylase of Bacillus subtilis. Biochim Biophys Acta. 1959 Jul;34:158–165. doi: 10.1016/0006-3002(59)90243-4. [DOI] [PubMed] [Google Scholar]
  55. Yuan T., Mietzner T. A., Montelaro R. C., Vogel H. J. Characterization of the calmodulin binding domain of SIV transmembrane glycoprotein by NMR and CD spectroscopy. Biochemistry. 1995 Aug 22;34(33):10690–10696. doi: 10.1021/bi00033a045. [DOI] [PubMed] [Google Scholar]
  56. Yuan T., Weljie A. M., Vogel H. J. Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding. Biochemistry. 1998 Mar 3;37(9):3187–3195. doi: 10.1021/bi9716579. [DOI] [PubMed] [Google Scholar]
  57. Zhang M., Fabian H., Mantsch H. H., Vogel H. J. Isotope-edited Fourier transform infrared spectroscopy studies of calmodulin's interaction with its target peptides. Biochemistry. 1994 Sep 13;33(36):10883–10888. doi: 10.1021/bi00202a006. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. 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]
  60. Zhang M., Vogel H. J. Determination of the side chain pKa values of the lysine residues in calmodulin. J Biol Chem. 1993 Oct 25;268(30):22420–22428. [PubMed] [Google Scholar]
  61. Zhang M., Vogel H. J. The calmodulin-binding domain of caldesmon binds to calmodulin in an alpha-helical conformation. Biochemistry. 1994 Feb 8;33(5):1163–1171. doi: 10.1021/bi00171a016. [DOI] [PubMed] [Google Scholar]
  62. Zhang M., Vogel H. J. Two-dimensional NMR studies of selenomethionyl calmodulin. J Mol Biol. 1994 Jun 17;239(4):545–554. doi: 10.1006/jmbi.1994.1393. [DOI] [PubMed] [Google Scholar]
  63. Zhang M., Yuan T., Aramini J. M., Vogel H. J. Interaction of calmodulin with its binding domain of rat cerebellar nitric oxide synthase. A multinuclear NMR study. J Biol Chem. 1995 Sep 8;270(36):20901–20907. doi: 10.1074/jbc.270.36.20901. [DOI] [PubMed] [Google Scholar]
  64. Zhang M., Yuan T., Vogel H. J. A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol. Protein Sci. 1993 Nov;2(11):1931–1937. doi: 10.1002/pro.5560021114. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES