Browse > Article
http://dx.doi.org/10.5352/JLS.2004.14.5.770

Functions of a-Tropomyosin Are Mainly Dependent upon the Local Structures of the Amino Terminus  

Cho, Young-Joon (Division of Life Science, Daegu University)
Publication Information
Journal of Life Science / v.14, no.5, 2004 , pp. 770-777 More about this Journal
Abstract
It has been previously reported that unacetylated a-tropomyosin(TM) produced in E. coli failed to bind to actin while acetylated muscle TM and Ala-Ser dipeptide fusion TM (AS-TM) bound well to actin. In order to determine the structural requirement of the amino terminus for high actin affinity, a recombinant tropomyosin (Ala-TM) that a single Ala residue was added to the amino terminus of Ala-TM was constructed, overexpressed, and purified from E. coli. Actin affinity of Ala-TM was 2.3$\times$$10^{6}$$M^{-1}$, whereas that of unacetylated TM was considerably lower than 0.1$\times$$10^{-6}$$M^{-1}$ indicating that addition of a single Ala residue to the amino terminus drastically increased, at least twenty times, actin affinity of TM. Ala-TM, however, bound to actin about three times weaker than acetylated TM and AS- TM, implying that the addition of an Ala residue was insufficient for complete restoration of high actin affinity. While Ala-TM, AS-TM, and muscle TM showed inhibition and activation of actomyosin Sl ATPase activity depending on myosin Sl concentration, the degree of inhibition and activation was different from each other. AS-TM exhibited the greatest inhibition of the ATPase at low Sl concentration, whereas the greatest activation of the ATPase was observed with muscle TM. These results, together with previous findings, strongly suggested that local structure of the amino terminus is the crucial functional determinant of TM.
Keywords
recombinant tropomyosins; actin binding; myosin Sl ATPase; N-acetylation;
Citations & Related Records
연도 인용수 순위
  • Reference
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   DOI   ScienceOn
2 Bharadwaj, S., S. E. Hitchcock-DeGregori, A. Thorburn, and G. L. Prasad. 2004. N-terminus is essential for tropomyosin functions. J. Biol. Chem. 279, 14039-14048   DOI   ScienceOn
3 Cho, Y. J. 2000. The carboxyl terminal amino acid residues glutamate276- threonine277 are important for actin affinity of the unacetylated smooth $\alpha$-tropomyosin. J. Biochem. Mol. Biol. 33, 531-536
4 Cho, Y. J. and S. E. Hitchcock-DeGregori. 1991. Relationship between alternatively spliced exons and functional domains in tropomyosin. Proc. Natl. Acad. Sci. USA. 88, 10153-10157   DOI   ScienceOn
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 Urbancikova, M. and S. E. Hitchcock-DeGregori. 1994. Requirement of amino-terminal modification for striated muscle alpha-tropomyosin function. J Biol Chem. 269, 24310-24315
7 White, H. D. 1982. Special instrumentation and techniques for kinetic studies of contractile systems. Methods Enzymol. 85(Pt B), 698-708   DOI
8 Williams, D. L., L. E. Greene and E. Eisenberg. 1988. Cooperative turning on myosin subfragment 1 adenosine triphosphatase activity by the troponin-tropomyosin-actin complex. Biochemistry 27, 6987-6993   DOI   ScienceOn
9 Winkelmann, D. A., H. Mekeel and I. Rayment. 1985. Packing analysis of crystalline myosin subfragment-1. Implication for the size and shape of myosin heads. J. Mol. Biol. 181, 487-501   DOI
10 Zot, A. S. and J. D. Potter. 1987. Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Ann. Rev. Biophys. Biophys. Chem. 16, 535-539   DOI
11 Polevoda, B. and F. Sherman. 2000. N$^{\alpha}$-terminal acetylation of eukaryotic proteins. J. Biol. Chem. 275, 36479-36482   DOI   ScienceOn
12 Pittenger, M. F., J. A. Kazzaz and D. M. Helfman. 1994. Functional properties of non-muscle tropomyosin isoforms. Curr. Opin. Cell Biol. 1, 96-104   DOI   ScienceOn
13 Pittenger, M. F., A. Kistler and D. M. Helfman. 1995. Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon. J. Cell Sci. 108, 3253-3265
14 Polevoda, B. and F. Sherman. (2003) N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins. J. Mol. Biol. 325, 595-622   DOI   ScienceOn
15 Polevoda, B., T. S. Cardillo, T. C. Doyle, G. S. Bedi and F. Sherman. 2003. Nat3p and Mdm20p are required for function of yeast NatB N$^{\alpha}$-terminal acetyltransferase and of actin and tropomyosin. J. Biol. Chem. 278, 30686-30697   DOI   ScienceOn
16 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
17 Lehrer, S. and Morris. 1982. Dual effects of tropomyosin and troponin-tropomyosin on actomyosin subfragment 1 ATPase. J. Biol. Chem. 257, 8073-8080
18 Sambrook. J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
19 Singer, J. and J. M. Shaw. 2003. Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin-actin interactions in budding yeast. Proc. Natl. Acad. Sci. 100, 7644-7649   DOI   ScienceOn
20 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685   DOI   ScienceOn
21 Maytum, R., M. A. Geeves and M. Konrad. 2000. Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification. Biochemistry. 39, 11913-11920   DOI   ScienceOn
22 Maytum, R., S. S. Lehrer and M. A. Geeves. 1999. Cooperativity and switching within the three-state model of muscle regulation. Biochemistry 38, 1102-1110   DOI   ScienceOn
23 Michele, D. E., F. P. Albayya and J. M. Metzger. 1999. Thin filament protein dynamics in fully differentiated adult cardiac myocytes: toward a model of sarcomere maintenance. J. Cell Biol. 145, 1483-1495   DOI
24 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
25 Greenfield N. J, T. Palm and S. E. Hitchcock-DeGregori. 2002. Structure and interactions of the carboxyl terminus of striated muscle alpha-tropomyosin: it is important to be flexible. Biophys J. 83, 2754-2766   DOI   ScienceOn
26 Moraczewska, M., K., Nicholson-Flynn and S. E. Hitchcock- DeGregori. 1999. The Ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding of F-actin in the open state. Biochemistry 38, 14885-15892   DOI   ScienceOn
27 Hitchcock-DeGregori, S. E., S. F. Lewis and T.M.-T. Chou. 1985. Tropomyosin lysine reactivities and relationship to coiled-coil structure. Biochemistry. 39, 11913-11920   DOI   ScienceOn
28 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
29 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   DOI
30 Greenfield, N., W. F. Stafford and S. E. Hitchcock- DeGregori. 1994. The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and $\alpha$$\alpha$-tropomyosin. Protein Sci. 3, 402-10   DOI   ScienceOn
31 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   DOI
32 Hitchcock-DeGregori, S. E., S. Mandala and G. A. Sachs. 1982. Changes in actin lysine reactivities during polymerization detected using a competitive labeling method. J. Biol. Chem. 257, 12573-12580
33 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
34 Heeley, D. H., L. B., Smillie and E. M. Lohmeier-Vogel. 1989. Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase. Biochem J. 258, 831-836
35 Hirel, P-H., J-M. Schmitter, P. Dessen, G. Fayat and S. Blanquet. 1989 Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. 86, 8247-8251   DOI   ScienceOn