Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Glutamine Residue at 276 of smooth muscle α-tropomyosin is primarily responsible for higher actin affinity

평활근 α-트로포마이오신 Gln276잔기의 액틴친화력에 대한 중요성

  • Jung, Sun-Ju (Department of Genetic Engineering, Daegu University) ;
  • Cho, Young-Joon (Department of Genetic Engineering, Daegu University)
  • 정선주 (대구대학교 자연과학대학 유전공학과) ;
  • 조영준 (대구대학교 자연과학대학 유전공학과)
  • Published : 2007.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

Previous reports indicated that the carboxyl terminal residues, glutamine276-threonine277 in particular, were important for actin affinity of the unacetylated smooth ${\alpha}-tropomyosin$. To determine the role of the glutamine and threonine residues in C-terminal region in actin binding, we constructed mutant striated muscle ${\alpha}-tropomyosin$ (TMs), in which these two residues were individually substituted. These mutant tropomyosins, designated TM18 (HT) and TM19 (QA), were overexpressed in E. coli as an either unacetylated form or Ala-Ser. (AS) dipeptide fusion form, and were analyzed F-actin affinity by cosedimentation. Unacetylated TM19 (QA) bound to actin approximately three times stronger than TM18 (HT) and much stronger than ST (HA). AS/TM19 (QA) showed four times stronger, in actin affinity than AS/ST (HA) while AS/TM14 (QT) bound to actin stronger to some extent than AS/TM18 (HT). These results suggested that the presence of Gln residue at 276 be primarily attributed to higher actin affinity of smooth ${\alpha}-tropomyosin$.

평활근 ${\alpha}$-트로포마이오신의 높은 액틴 친화력은 아미노산 잔기 Gln276 및 Thr277에 기인한다는 이전 보고에 따라, 2 잔기 중 어느 잔기가 액틴 친화력에 더 중요한 가를 알아보기 위하여 골격근 트로포마이오신의 His 혹은 Ala 단일 잔기를 각각 Gln 혹은 Thr으로 치환한 돌연변이 트로포마이오신을 제작하여 대장균에서 대량발현 시킨 후 정제하여 액틴 결합력을 측정하였다. 비록 비아세틸화된 트로포마이오신의 경우 Gln 및 Thr 잔기가 최고 액틴친화력을 위해 모두 필요하나, 돌연변이 트로포마이신 중 Gln 잔기를 가진 돌연변이 트로포마이오신들이 다른 돌연변이 트로포마이오신들에 비하여 3에서 4배 높은 액틴친화력을 보였다. 이러한 결과는 평활근 ${\alpha}$-트로포마이오신의 높은 액틴 친화력은 Thr277 잔기보다 Gln276 잔기에 주로 기인한다는 것을 의미한다.

Keywords

References

  1. Bradford, M. M. 1976. A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  2. Cho, Y. and S. E. Hitchcock-DeGregori. 1991. Relationship between alternatively spliced exons and functional domains in tropomyosin. Proc. Natl. Acad. Sci. USA 88, 10153-10157 https://doi.org/10.1073/pnas.88.22.10153
  3. Cho, Y. J. 2000. The carboxyl terminal amino acid residues glutamine 276 - threonine 277 are important for actin affinity of the unacetylated smooth $\alpha$-tropomyosin. J. Biochem. Mol. Biol. 33, 531-536
  4. Cho, Y. J. 2004 Functions of ${\alpha}$-tropomyosin are mainly dependent upon the local structures of the amino terminus. J. Life Science 14, 770-777 https://doi.org/10.5352/JLS.2004.14.5.770
  5. Cho, Y. J., J. Liu and S. E. Hitchcock-DeGregori. 1990. The amino terminus of muscle tropomyosin is a major determinant for function. J. Biol. Chem. 265, 538-545
  6. Gaffin, R. D., C. W. Tong, C. Z. Zaweija, T. E. Hewett, R. Klevitsky, J. Robbins and M. Muthuchamy. 2004. Charged residue alterations in the inner-core domain and carboxy-terminus of ${\alpha}$-tropomyosin differentially affect mouse cardiac muscle contractility. J. Physiol. 561, 777-791 https://doi.org/10.1113/jphysiol.2004.070631
  7. Gaffin, R. D., K Gokulan, J. C. Sacchettini, C. W. Tong, T. E. Hewett, R. Klevitsky, J. Robbins and M. Muthuchamy. 2004. Charged residue changes in the carboxy-terminus of ${\alpha}$-tropomyosin alter mouse cardiac muscle contractility. J. Physiol. 556, 531-543 https://doi.org/10.1113/jphysiol.2003.058487
  8. Greenfield N. J., T. Palm and S. E. Hitchcock-DeGregori. 2002. Structure and interactions of the carboxyl terminus of striated muscle a-tropomyosin: it is important to be flexible. Biophys. J. 83, 2754-2766 https://doi.org/10.1016/S0006-3495(02)75285-5
  9. Greenfield, N. J. and S. E. Hitchcock-DeGregori. 1995. The stability of tropomyosin, a two-stranded coiled-coil protein, is primarily a function of the hydrophobicity of residues at the helix-helix interface. Biochemistry 34, 16797-16805 https://doi.org/10.1021/bi00051a030
  10. Greenfield, N. J., G. V .T. Swapna, Y. Huang, T. Palm, S. Graboski, G. T. Montelione and S. E. Hitchcock-DeGregori. 2003. The structure of the carboxyl terminus of striated ${\alpha}$-tropomyosin in solution reveals an unusual parallel arrangement of interacting ${\alpha}$-helices. Biochemistry 42, 614-619 https://doi.org/10.1021/bi026989e
  11. Gunning, P. W., G. Schevzov, A. J. Kee and E. C. Hardeman. 2005. Tropomyosin isoforrns: dividing rods for actin cytoskeleton function. Trends Cell Biol. 15, 333-341 https://doi.org/10.1016/j.tcb.2005.04.007
  12. Hammell, R. and S. E. Hitchcock-DeGregori. 1996. Mapping the functional domains within the carboxyl terminus of alpha-tropomyosin encoded by the alternatively spliced ninth exon. J. Biol. Chem. 271, 4236-4242 https://doi.org/10.1074/jbc.271.8.4236
  13. Heald, R. W. and S. E. Hitchcock-DeGregori. 1988. The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin J. Biol. Chem. 263, 5254-5259
  14. Jagatheesan, G., S. Rajan, N. Petrashevskaya, A. Schwartz, G. Bolvin, S. Vahebi, P. DeTombe, R. J. Solaro, E. Labitzke, G. Hillard and D. W. Wieczorek. 2003. Functional importance of the carboxyl-terminal region of striated muscle tropomyosin. J. Biol. Chem. 278, 23204-23211 https://doi.org/10.1074/jbc.M303073200
  15. Jung, S. J., S. M. Seo, K. H. Suh, J. S. Yang and Y. J. Cho. 2001. Effect of three amino acid residues at the carboxyl terminus in unacetylated ${\alpha}$-tropomyosin on actin affinity. J. Life Science 11, 1-6
  16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  17. McLachlan, A. D. and M. Stewart. 1975. Tropomyosin coiled-coil interactions: evidence for an unstaggered structure. J. Mol Biol. 98, 293-304 https://doi.org/10.1016/S0022-2836(75)80119-7
  18. Monteiro, P. B., R. C. Lataro, J. A. Ferro and F. d. C. Reinach. 1994. Functional ${\alpha}$-tropomyosin produced in Escherichia coli. A dipeptide extension can substitute the amino terminal acetyl group. J. Biol. Chem. 269, 10461-10466
  19. Moraczewska, J., K. Nicholson-Flynn and S. E. Hitchcock-DeGregori. 1999. The ends of tropomyosin are major determinants of actin affinity and myosin subfragment I-induced binding of F-actin in open state. Biochemistry 38, 15885-15892 https://doi.org/10.1021/bi991816j
  20. Palm, T., N. J. Greenfield and S. E. Hitchcock-DeGregori 2003. Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes. Biophys. J. 84, 3181-3189 https://doi.org/10.1016/S0006-3495(03)70042-3
  21. Palm, T., S. Graboski, S. E. Hitchcock-DeGregori and N. J. Greenfield. 2001. Disease-causing mutations in cardiac troponin T: identification of a critical tropomyosin-binding region. Biophys. J. 81, 2827-2837 https://doi.org/10.1016/S0006-3495(01)75924-3
  22. Perry, S. V. 2001. Vertebrate tropomyosin: distribution, properties and function. J. Muscle Res. Cell Motil. 22, 5-49 https://doi.org/10.1023/A:1010303732441
  23. Pittenger, M. F., J. A. Kazzaz and D. M. Helfman. 1994. Functional properties of non-muscle tropomyosin isoforms. Curr. Opin. Cell Biol. 6, 96-104 https://doi.org/10.1016/0955-0674(94)90122-8
  24. Ruiz-Opazo, N. and B. Nadal-Ginard. 1987. ${\alpha}$-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms. J. Biol. Chem. 262, 4755-4765
  25. Tobacman L. S. 1996. Thin filament-mediated regulation of cardiac contraction. Annu. Rev. Physiol. 58, 447-81 https://doi.org/10.1146/annurev.ph.58.030196.002311
  26. Urbancikova, M. and S. E. Hltchcock-DeGregori. 1994. Requirement of amino-terminal modification for striated muscle ${\alpha}$-tropomyosin function. J. Biol. Chem. 269, 24310-24315
  27. Yang, Y. Z., E. D. Korn and E. Eisenberg. 1979. Cooperative binding of tropomyosin to muscle and Acanthamoeba actin. J. Biol. Chem. 254, 2084-2088