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. 2002 Sep;83(3):1455–1464. doi: 10.1016/S0006-3495(02)73916-7

Dynamic light scattering study of calmodulin-target peptide complexes.

Andriyka L Papish 1, Leslie W Tari 1, Hans J Vogel 1
PMCID: PMC1302244  PMID: 12202371

Abstract

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.

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Selected References

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  1. 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]
  2. 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]
  3. Brokx R. D., Lopez M. M., Vogel H. J., Makhatadze G. I. Energetics of target peptide binding by calmodulin reveals different modes of binding. J Biol Chem. 2001 Jan 29;276(17):14083–14091. doi: 10.1074/jbc.M011026200. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Czuryło E. A., Hellweg T., Eimer W., Dabrowska R. The size and shape of caldesmon and its fragments in solution studied by dynamic light scattering and hydrodynamic model calculations. Biophys J. 1997 Feb;72(2 Pt 1):835–842. doi: 10.1016/s0006-3495(97)78717-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fabian H., Yuan T., Vogel H. J., Mantsch H. H. Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy. Eur Biophys J. 1996;24(4):195–201. doi: 10.1007/BF00205100. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gomes A. V., Barnes J. A., Vogel H. J. Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin. Arch Biochem Biophys. 2000 Jul 1;379(1):28–36. doi: 10.1006/abbi.2000.1827. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Ikura M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci. 1996 Jan;21(1):14–17. [PubMed] [Google Scholar]
  11. 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]
  12. Izumi Y., Kuwamoto S., Jinbo Y., Yoshino H. Increase in the molecular weight and radius of gyration of apocalmodulin induced by binding of target peptide: evidence for complex formation. FEBS Lett. 2001 Apr 20;495(1-2):126–130. doi: 10.1016/s0014-5793(01)02375-4. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Kataoka M., Head J. F., Vorherr T., Krebs J., Carafoli E. Small-angle X-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane Ca2+ pump. Biochemistry. 1991 Jun 25;30(25):6247–6251. doi: 10.1021/bi00239a024. [DOI] [PubMed] [Google Scholar]
  16. Kranz James K., Lee Eun K., Nairn Angus C., Wand A. Joshua. A direct test of the reductionist approach to structural studies of calmodulin activity: relevance of peptide models of target proteins. J Biol Chem. 2002 Mar 19;277(19):16351–16354. doi: 10.1074/jbc.C200139200. [DOI] [PubMed] [Google Scholar]
  17. Krueger J. K., Gallagher S. C., Wang C. A., Trewhella J. Calmodulin remains extended upon binding to smooth muscle caldesmon: a combined small-angle scattering and fourier transform infrared spectroscopy study. Biochemistry. 2000 Apr 11;39(14):3979–3987. doi: 10.1021/bi992638x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Murphy RM. Static and dynamic light scattering of biological macromolecules: what can we learn? Curr Opin Biotechnol. 1997 Feb 1;8(1):25–30. doi: 10.1016/s0958-1669(97)80153-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Schumacher M. A., Rivard A. F., Bächinger H. P., Adelman J. P. Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin. Nature. 2001 Apr 26;410(6832):1120–1124. doi: 10.1038/35074145. [DOI] [PubMed] [Google Scholar]
  24. Seaton B. A., Head J. F., Engelman D. M., Richards F. M. Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle X-ray scattering. Biochemistry. 1985 Nov 19;24(24):6740–6743. doi: 10.1021/bi00345a002. [DOI] [PubMed] [Google Scholar]
  25. Squire P. G., Himmel M. E. Hydrodynamics and protein hydration. Arch Biochem Biophys. 1979 Aug;196(1):165–177. doi: 10.1016/0003-9861(79)90563-0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Trewhella J., Blumenthal D. K., Rokop S. E., Seeger P. A. Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase. Biochemistry. 1990 Oct 9;29(40):9316–9324. doi: 10.1021/bi00492a003. [DOI] [PubMed] [Google Scholar]
  28. Trewhella J. Insights into biomolecular function from small-angle scattering. Curr Opin Struct Biol. 1997 Oct;7(5):702–708. doi: 10.1016/s0959-440x(97)80081-4. [DOI] [PubMed] [Google Scholar]
  29. Trewhella Jill, Krueger Joanna K. Small-angle solution scattering reveals information on conformational dynamics in calcium-binding proteins and in their interactions with regulatory targets. Methods Mol Biol. 2002;173:137–159. doi: 10.1385/1-59259-184-1:137. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Wang E., Zhuang S., Kordowska J., Grabarek Z., Wang C. L. Calmodulin binds to caldesmon in an antiparallel manner. Biochemistry. 1997 Dec 2;36(48):15026–15034. doi: 10.1021/bi963075h. [DOI] [PubMed] [Google Scholar]
  33. Yajima H., Yamamoto H., Nagaoka M., Nakazato K., Ishii T., Niimura N. Small-angle neutron scattering and dynamic light scattering studies of N- and C-terminal fragments of ovotransferrin. Biochim Biophys Acta. 1998 Jun 5;1381(1):68–76. doi: 10.1016/s0304-4165(98)00014-2. [DOI] [PubMed] [Google Scholar]
  34. Yoshino H., Izumi Y., Sakai K., Takezawa H., Matsuura I., Maekawa H., Yazawa M. Solution X-ray scattering data show structural differences between yeast and vertebrate calmodulin: implications for structure/function. Biochemistry. 1996 Feb 20;35(7):2388–2393. doi: 10.1021/bi952121v. [DOI] [PubMed] [Google Scholar]
  35. Yoshino H., Wakita M., Izumi Y. Calcium-dependent changes in structure of calmodulin with substance P. J Biol Chem. 1993 Jun 5;268(16):12123–12128. [PubMed] [Google Scholar]
  36. 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]
  37. Yuan T., Ouyang H., Vogel H. J. Surface exposure of the methionine side chains of calmodulin in solution. A nitroxide spin label and two-dimensional NMR study. J Biol Chem. 1999 Mar 26;274(13):8411–8420. doi: 10.1074/jbc.274.13.8411. [DOI] [PubMed] [Google Scholar]
  38. Yuan T., Tencza S., Mietzner T. A., Montelaro R. C., Vogel H. J. Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41. Biopolymers. 2001 Jan;58(1):50–62. doi: 10.1002/1097-0282(200101)58:1<50::AID-BIP60>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  39. Yuan T., Vogel H. J. Calcium-calmodulin-induced dimerization of the carboxyl-terminal domain from petunia glutamate decarboxylase. A novel calmodulin-peptide interaction motif. J Biol Chem. 1998 Nov 13;273(46):30328–30335. doi: 10.1074/jbc.273.46.30328. [DOI] [PubMed] [Google Scholar]
  40. Yuan T., Walsh M. P., Sutherland C., Fabian H., Vogel H. J. Calcium-dependent and -independent interactions of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with calmodulin. Biochemistry. 1999 Feb 2;38(5):1446–1455. doi: 10.1021/bi9816453. [DOI] [PubMed] [Google Scholar]
  41. Zarutskie J. A., Sato A. K., Rushe M. M., Chan I. C., Lomakin A., Benedek G. B., Stern L. J. A conformational change in the human major histocompatibility complex protein HLA-DR1 induced by peptide binding. Biochemistry. 1999 May 4;38(18):5878–5887. doi: 10.1021/bi983048m. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Zhang M., Vogel H. J. Characterization of the calmodulin-binding domain of rat cerebellar nitric oxide synthase. J Biol Chem. 1994 Jan 14;269(2):981–985. [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Zhou N., Yuan T., Mak A. S., Vogel H. J. NMR studies of caldesmon-calmodulin interactions. Biochemistry. 1997 Mar 11;36(10):2817–2825. doi: 10.1021/bi9625713. [DOI] [PubMed] [Google Scholar]

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