A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization

Adriana A. Paulucci, Angela M. Katsuyama, Aurea D. Sousa, Chuck S. Farah

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as α-amino acetylation of the N-terminus of the muscle protein. Nα- acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.

    Original languageEnglish (US)
    Pages (from-to)589-600
    Number of pages12
    JournalEuropean Journal of Biochemistry
    Volume271
    Issue number3
    DOIs
    StatePublished - Feb 2004

    Fingerprint

    Tropomyosin
    Polymerization
    Acetylation
    Fusion reactions
    Ionic strength
    Osmolar Concentration
    Proteins
    Muscle Proteins
    Recombinant Proteins
    Escherichia coli
    Actins
    Head
    Amino Acids

    Keywords

    • Circular dichroism
    • Head-to-tail interaction
    • Protein folding
    • Protein polymerization
    • Tropomyosin

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization. / Paulucci, Adriana A.; Katsuyama, Angela M.; Sousa, Aurea D.; Farah, Chuck S.

    In: European Journal of Biochemistry, Vol. 271, No. 3, 02.2004, p. 589-600.

    Research output: Contribution to journalArticle

    Paulucci, Adriana A. ; Katsuyama, Angela M. ; Sousa, Aurea D. ; Farah, Chuck S. / A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization. In: European Journal of Biochemistry. 2004 ; Vol. 271, No. 3. pp. 589-600.
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    abstract = "Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as α-amino acetylation of the N-terminus of the muscle protein. Nα- acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.",
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    AU - Farah, Chuck S.

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    AB - Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as α-amino acetylation of the N-terminus of the muscle protein. Nα- acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.

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