title: N-Terminal Hypervariable Region of Muscle Type Isoforms of Troponin T Differentially Modulates the Affinity of Tropomyosin-Binding Site 1.
abstract: The troponin complex plays a central role in the contraction and relaxation of striated muscle by enacting Ca²⁺-regulated allosteric changes in the sarcomeric thin filaments. The troponin T subunit (TnT) contains two binding sites for tropomyosin (Tm) and is responsible for anchoring the troponin complex to the thin filament. While the amino acid sequences of the regions containing the Tm-binding sites are highly conserved among the three muscle type isoforms of TnT, previous studies have observed significant discrepancies in the affinity of Tm-binding site 1 in the chymotryptic fragment T1 of different TnT isoforms. Here we cross-examined the Tm-binding affinity of TnT isoforms and molecular engineered fragments using affinity chromatography and microplate protein binding assays to investigate the effects of the evolutionarily diverged N-terminal region that is significantly variable among muscle type isoforms. The results demonstrated that the C-terminal T2 fragment of TnT containing the Tm-binding site 2 had similarly high affinity across isoforms. In the absence of the N-terminal variable region, Tm-binding site 1 in the conserved middle region of TnT also exhibited high intrinsic affinity. The presence of isoform specific N-terminal variable region differentially reduced the binding affinity of TnT for Tm, primarily at binding site 1 in the middle region. These findings indicate that the N-terminal variable region of TnT plays a pivotal role in the functional difference of muscle type-specific isoforms and the developmental and pathogenic splice variants by modulating the interaction with Tm during Ca²⁺ regulation of cardiac and skeletal muscle contraction and relaxation.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, United States.
authors.forename: Chinthaka
authors.initials: C
authors.lastname: Amarasinghe
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, United States.
authors.forename: J-P
authors.initials: JP
authors.lastname: Jin
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism*; D020107:Troponin T / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins; D014335:Tropomyosin; D020107:Troponin T