Expression, zinc-affinity purification, and characterization of a novel metal-binding cluster in troponin T: Metal-stabilized α-helical structure and effects of the NH2-terminal variable region on the conformation of intact troponin T and its association with tropomyosin

O. Ogut, J. P. Jin

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

A repeating metal-binding (Cu2+ > Ni2+ ≃ Zn2+ ≃ Co2+) sequence [HE/AEAH]4 (Tx) has been recently identified in the NH2-terminal variable region of troponin T (TnT) isoforms specifically expressed in the breast but not leg muscles of the avian orders of Galliformes and Craciformes [Jin, J.- P., and Smillie, L. B. (1994) FEBS Lett. 341, 135-140]. In the present study, two expression plasmids were constructed to produce chicken TnT1 NH2- terminal fragments of 47 (N47) or 165 (N165) amino acids containing the Tx metal-binding cluster. The recombinant protein/peptide was expressed in Escherichia coli BL21(DE3)pLysS and purified by a highly effective Zn2+- affinity chromatography method. Amino acid analyses, NH2-terminal peptide sequencing, mass spectrometry and immunological identification confirmed the authenticity of the genetically engineered TnT fragments. In the presence of 2,2,2-trifluoroethanol, transition metals had significant effects on the secondary structure of TnT fragment N47, as shown by circular dichroism. N165 in non-denaturing buffer demonstrated α-helical content comparable to previous data from rabbit fast skeletal TnT fragment T1. Zn2+-binding avidity of the metal-binding TnT and its fragments demonstrated tertiary relationships between the NH2-terminal variable region and the COOH-terminal segment of the intact TnT protein. Solid-phase protein-binding assays established that Zn2+-binding to the Tx cluster induces epitopic structure changes in this NH2-terminal segment, further affecting other epitopic structures of intact TnT as well as the function of TnT's tropomyosin binding-sites. The results demonstrate that metal ion-binding to the Tx cluster reconfigures the overall conformation of TnT through structural relationships between the NH2-terminal variable region and other domains of the intact TnT molecule. Accordingly, the developmental and/or muscle type specific NH2-terminal structure of TnT isoforms may modulate the Ca2+- activation of muscle contraction.

Original languageEnglish (US)
Pages (from-to)16581-16590
Number of pages10
JournalBiochemistry
Volume35
Issue number51
DOIs
StatePublished - 1996
Externally publishedYes

Fingerprint

Tropomyosin
Troponin T
Purification
Conformations
Zinc
Metals
Association reactions
Muscle
Galliformes
Protein Isoforms
Trifluoroethanol
Affinity chromatography
Amino Acids
Muscles
Peptides
Muscle Contraction
Circular Dichroism
Affinity Chromatography
Recombinant Proteins
Protein Binding

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{c8fd648a9a314e2fbdb48d957fc645d0,
title = "Expression, zinc-affinity purification, and characterization of a novel metal-binding cluster in troponin T: Metal-stabilized α-helical structure and effects of the NH2-terminal variable region on the conformation of intact troponin T and its association with tropomyosin",
abstract = "A repeating metal-binding (Cu2+ > Ni2+ ≃ Zn2+ ≃ Co2+) sequence [HE/AEAH]4 (Tx) has been recently identified in the NH2-terminal variable region of troponin T (TnT) isoforms specifically expressed in the breast but not leg muscles of the avian orders of Galliformes and Craciformes [Jin, J.- P., and Smillie, L. B. (1994) FEBS Lett. 341, 135-140]. In the present study, two expression plasmids were constructed to produce chicken TnT1 NH2- terminal fragments of 47 (N47) or 165 (N165) amino acids containing the Tx metal-binding cluster. The recombinant protein/peptide was expressed in Escherichia coli BL21(DE3)pLysS and purified by a highly effective Zn2+- affinity chromatography method. Amino acid analyses, NH2-terminal peptide sequencing, mass spectrometry and immunological identification confirmed the authenticity of the genetically engineered TnT fragments. In the presence of 2,2,2-trifluoroethanol, transition metals had significant effects on the secondary structure of TnT fragment N47, as shown by circular dichroism. N165 in non-denaturing buffer demonstrated α-helical content comparable to previous data from rabbit fast skeletal TnT fragment T1. Zn2+-binding avidity of the metal-binding TnT and its fragments demonstrated tertiary relationships between the NH2-terminal variable region and the COOH-terminal segment of the intact TnT protein. Solid-phase protein-binding assays established that Zn2+-binding to the Tx cluster induces epitopic structure changes in this NH2-terminal segment, further affecting other epitopic structures of intact TnT as well as the function of TnT's tropomyosin binding-sites. The results demonstrate that metal ion-binding to the Tx cluster reconfigures the overall conformation of TnT through structural relationships between the NH2-terminal variable region and other domains of the intact TnT molecule. Accordingly, the developmental and/or muscle type specific NH2-terminal structure of TnT isoforms may modulate the Ca2+- activation of muscle contraction.",
author = "O. Ogut and Jin, {J. P.}",
year = "1996",
doi = "10.1021/bi961712y",
language = "English (US)",
volume = "35",
pages = "16581--16590",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
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TY - JOUR

T1 - Expression, zinc-affinity purification, and characterization of a novel metal-binding cluster in troponin T

T2 - Metal-stabilized α-helical structure and effects of the NH2-terminal variable region on the conformation of intact troponin T and its association with tropomyosin

AU - Ogut, O.

AU - Jin, J. P.

PY - 1996

Y1 - 1996

N2 - A repeating metal-binding (Cu2+ > Ni2+ ≃ Zn2+ ≃ Co2+) sequence [HE/AEAH]4 (Tx) has been recently identified in the NH2-terminal variable region of troponin T (TnT) isoforms specifically expressed in the breast but not leg muscles of the avian orders of Galliformes and Craciformes [Jin, J.- P., and Smillie, L. B. (1994) FEBS Lett. 341, 135-140]. In the present study, two expression plasmids were constructed to produce chicken TnT1 NH2- terminal fragments of 47 (N47) or 165 (N165) amino acids containing the Tx metal-binding cluster. The recombinant protein/peptide was expressed in Escherichia coli BL21(DE3)pLysS and purified by a highly effective Zn2+- affinity chromatography method. Amino acid analyses, NH2-terminal peptide sequencing, mass spectrometry and immunological identification confirmed the authenticity of the genetically engineered TnT fragments. In the presence of 2,2,2-trifluoroethanol, transition metals had significant effects on the secondary structure of TnT fragment N47, as shown by circular dichroism. N165 in non-denaturing buffer demonstrated α-helical content comparable to previous data from rabbit fast skeletal TnT fragment T1. Zn2+-binding avidity of the metal-binding TnT and its fragments demonstrated tertiary relationships between the NH2-terminal variable region and the COOH-terminal segment of the intact TnT protein. Solid-phase protein-binding assays established that Zn2+-binding to the Tx cluster induces epitopic structure changes in this NH2-terminal segment, further affecting other epitopic structures of intact TnT as well as the function of TnT's tropomyosin binding-sites. The results demonstrate that metal ion-binding to the Tx cluster reconfigures the overall conformation of TnT through structural relationships between the NH2-terminal variable region and other domains of the intact TnT molecule. Accordingly, the developmental and/or muscle type specific NH2-terminal structure of TnT isoforms may modulate the Ca2+- activation of muscle contraction.

AB - A repeating metal-binding (Cu2+ > Ni2+ ≃ Zn2+ ≃ Co2+) sequence [HE/AEAH]4 (Tx) has been recently identified in the NH2-terminal variable region of troponin T (TnT) isoforms specifically expressed in the breast but not leg muscles of the avian orders of Galliformes and Craciformes [Jin, J.- P., and Smillie, L. B. (1994) FEBS Lett. 341, 135-140]. In the present study, two expression plasmids were constructed to produce chicken TnT1 NH2- terminal fragments of 47 (N47) or 165 (N165) amino acids containing the Tx metal-binding cluster. The recombinant protein/peptide was expressed in Escherichia coli BL21(DE3)pLysS and purified by a highly effective Zn2+- affinity chromatography method. Amino acid analyses, NH2-terminal peptide sequencing, mass spectrometry and immunological identification confirmed the authenticity of the genetically engineered TnT fragments. In the presence of 2,2,2-trifluoroethanol, transition metals had significant effects on the secondary structure of TnT fragment N47, as shown by circular dichroism. N165 in non-denaturing buffer demonstrated α-helical content comparable to previous data from rabbit fast skeletal TnT fragment T1. Zn2+-binding avidity of the metal-binding TnT and its fragments demonstrated tertiary relationships between the NH2-terminal variable region and the COOH-terminal segment of the intact TnT protein. Solid-phase protein-binding assays established that Zn2+-binding to the Tx cluster induces epitopic structure changes in this NH2-terminal segment, further affecting other epitopic structures of intact TnT as well as the function of TnT's tropomyosin binding-sites. The results demonstrate that metal ion-binding to the Tx cluster reconfigures the overall conformation of TnT through structural relationships between the NH2-terminal variable region and other domains of the intact TnT molecule. Accordingly, the developmental and/or muscle type specific NH2-terminal structure of TnT isoforms may modulate the Ca2+- activation of muscle contraction.

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