Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1996 Oct;71(4):1914–1919. doi: 10.1016/S0006-3495(96)79390-6

Intermolecular coupling between loop 38-52 and the C-terminus in actin filaments.

E Kim 1, E Reisler 1
PMCID: PMC1233658  PMID: 8889166

Abstract

The recently reported structural connectivity in F-actin between the DNase I binding loop on actin (residues 38-52) and the C-terminus region was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fluorescence of dansyl ethylenediamine (DED) attached to Gin-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleimide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1:9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers (1:4) of DED-actin and N-ethylmaleimide-actin. These results show intermolecular coupling between loop 38-52 and the C-terminus in F-actin. Consistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-actin but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase activity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive changes in actin structure and that the perturbation of loop 38-52 environment results from changes in the intermolecular contacts in F-actin.

Full text

PDF
1916

Selected References

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

  1. Adams S. B., Reisler E. Sequence 18-29 on actin: antibody and spectroscopic probing of conformational changes. Biochemistry. 1994 Dec 6;33(48):14426–14433. doi: 10.1021/bi00252a008. [DOI] [PubMed] [Google Scholar]
  2. Crosbie R. H., Miller C., Cheung P., Goodnight T., Muhlrad A., Reisler E. Structural connectivity in actin: effect of C-terminal modifications on the properties of actin. Biophys J. 1994 Nov;67(5):1957–1964. doi: 10.1016/S0006-3495(94)80678-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Drabikowski W., Lehrer S., Nagy B., Gergely J. Loss of Cu2+-binding to actin upon removal of the C-terminal phenylalanine by carboxypeptidase A. Arch Biochem Biophys. 1977 May;181(1):359–361. doi: 10.1016/0003-9861(77)90515-x. [DOI] [PubMed] [Google Scholar]
  4. Drewes G., Faulstich H. Cooperative effects on filament stability in actin modified at the C-terminus by substitution or truncation. Eur J Biochem. 1993 Feb 15;212(1):247–253. doi: 10.1111/j.1432-1033.1993.tb17656.x. [DOI] [PubMed] [Google Scholar]
  5. Drummond D. R., Hennessey E. S., Sparrow J. C. The binding of mutant actins to profilin, ATP and DNase I. Eur J Biochem. 1992 Oct 1;209(1):171–179. doi: 10.1111/j.1432-1033.1992.tb17274.x. [DOI] [PubMed] [Google Scholar]
  6. Egelman E. H., Orlova A. New insights into actin filament dynamics. Curr Opin Struct Biol. 1995 Apr;5(2):172–180. doi: 10.1016/0959-440x(95)80072-7. [DOI] [PubMed] [Google Scholar]
  7. Fievez S., Carlier M. F. Conformational changes in subdomain-2 of G-actin upon polymerization into F-actin and upon binding myosin subfragment-1. FEBS Lett. 1993 Jan 25;316(2):186–190. doi: 10.1016/0014-5793(93)81212-i. [DOI] [PubMed] [Google Scholar]
  8. Godfrey J. E., Harrington W. F. Self-association in the myosin system at high ionic strength. I. Sensitivity of the interaction to pH and ionic environment. Biochemistry. 1970 Feb 17;9(4):886–893. doi: 10.1021/bi00806a025. [DOI] [PubMed] [Google Scholar]
  9. Jacobson G. R., Rosenbusch J. P. ATP binding to a protease-resistant core of actin. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2742–2746. doi: 10.1073/pnas.73.8.2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kasprzak A. A., Takashi R., Morales M. F. Orientation of actin monomer in the F-actin filament: radial coordinate of glutamine-41 and effect of myosin subfragment 1 binding on the monomer orientation. Biochemistry. 1988 Jun 14;27(12):4512–4522. doi: 10.1021/bi00412a044. [DOI] [PubMed] [Google Scholar]
  11. Khaitlina S. Y., Moraczewska J., Strzelecka-Gołaszewska H. The actin/actin interactions involving the N-terminus of the DNase-I-binding loop are crucial for stabilization of the actin filament. Eur J Biochem. 1993 Dec 15;218(3):911–920. doi: 10.1111/j.1432-1033.1993.tb18447.x. [DOI] [PubMed] [Google Scholar]
  12. Kim E., Miller C. J., Motoki M., Seguro K., Muhlrad A., Reisler E. Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41. Biophys J. 1996 Mar;70(3):1439–1446. doi: 10.1016/S0006-3495(96)79703-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kim E., Motoki M., Seguro K., Muhlrad A., Reisler E. Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41. Biophys J. 1995 Nov;69(5):2024–2032. doi: 10.1016/S0006-3495(95)80072-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kouyama T., Mihashi K. Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem. 1981;114(1):33–38. [PubMed] [Google Scholar]
  15. Kuznetsova I., Antropova O., Turoverov K., Khaitlina S. Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I-binding loop: energy transfer from tryptophan to AEDANS. FEBS Lett. 1996 Mar 25;383(1-2):105–108. doi: 10.1016/0014-5793(96)00238-4. [DOI] [PubMed] [Google Scholar]
  16. Lehrer S. S., Nagy B., Gergely J. The binding of Cu 2+ to actin without loss of polymerizability: the involvement of the rapidly reacting -SH group. Arch Biochem Biophys. 1972 May;150(1):164–174. doi: 10.1016/0003-9861(72)90023-9. [DOI] [PubMed] [Google Scholar]
  17. Lorenz M., Popp D., Holmes K. C. Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. J Mol Biol. 1993 Dec 5;234(3):826–836. doi: 10.1006/jmbi.1993.1628. [DOI] [PubMed] [Google Scholar]
  18. Muhlrad A., Cheung P., Phan B. C., Miller C., Reisler E. Dynamic properties of actin. Structural changes induced by beryllium fluoride. J Biol Chem. 1994 Apr 22;269(16):11852–11858. [PubMed] [Google Scholar]
  19. Orlova A., Egelman E. H. A conformational change in the actin subunit can change the flexibility of the actin filament. J Mol Biol. 1993 Jul 20;232(2):334–341. doi: 10.1006/jmbi.1993.1393. [DOI] [PubMed] [Google Scholar]
  20. Orlova A., Egelman E. H. Structural basis for the destabilization of F-actin by phosphate release following ATP hydrolysis. J Mol Biol. 1992 Oct 20;227(4):1043–1053. doi: 10.1016/0022-2836(92)90520-t. [DOI] [PubMed] [Google Scholar]
  21. Orlova A., Egelman E. H. Structural dynamics of F-actin: I. Changes in the C terminus. J Mol Biol. 1995 Feb 3;245(5):582–597. doi: 10.1006/jmbi.1994.0048. [DOI] [PubMed] [Google Scholar]
  22. Orlova A., Prochniewicz E., Egelman E. H. Structural dynamics of F-actin: II. Cooperativity in structural transitions. J Mol Biol. 1995 Feb 3;245(5):598–607. doi: 10.1006/jmbi.1994.0049. [DOI] [PubMed] [Google Scholar]
  23. Owen C., DeRosier D. A 13-A map of the actin-scruin filament from the limulus acrosomal process. J Cell Biol. 1993 Oct;123(2):337–344. doi: 10.1083/jcb.123.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schwyter D. H., Kron S. J., Toyoshima Y. Y., Spudich J. A., Reisler E. Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin. J Cell Biol. 1990 Aug;111(2):465–470. doi: 10.1083/jcb.111.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schwyter D., Phillips M., Reisler E. Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1. Biochemistry. 1989 Jul 11;28(14):5889–5895. doi: 10.1021/bi00440a027. [DOI] [PubMed] [Google Scholar]
  26. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  27. Strzelecka-Gołaszewska H., Moraczewska J., Khaitlina S. Y., Mossakowska M. Localization of the tightly bound divalent-cation-dependent and nucleotide-dependent conformation changes in G-actin using limited proteolytic digestion. Eur J Biochem. 1993 Feb 1;211(3):731–742. doi: 10.1111/j.1432-1033.1993.tb17603.x. [DOI] [PubMed] [Google Scholar]
  28. Tirion M. M., ben-Avraham D., Lorenz M., Holmes K. C. Normal modes as refinement parameters for the F-actin model. Biophys J. 1995 Jan;68(1):5–12. doi: 10.1016/S0006-3495(95)80156-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weeds A. G., Pope B. Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility. J Mol Biol. 1977 Apr;111(2):129–157. doi: 10.1016/s0022-2836(77)80119-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES