Conformational modulation of slow skeletal muscle troponin T by an NH(2)-terminal metal-binding extension.
Troponin T (TnT) is an essential element in the thin filament Ca(2+)-regulatory system controlling striated muscle contraction. Alternative RNA splicing generates developmental and muscle type-specific TnT isoforms differing in the hypervariable NH(2)-terminal region. Using avian fast skeletal muscle TnT containing a metal-binding segment, we have demonstrated a role of the NH(2)-terminal domain in modulating the conformation of TnT (Wang J and Jin JP. Biochemistry 37: 14519-14528, 1998). To further investigate the structure-function relationship of TnT, the present study constructed and characterized a recombinant protein in which the metal-binding peptide present in avian fast skeletal muscle TnT was fused to the NH(2) terminus of mouse slow skeletal muscle TnT. Metal ion or monoclonal antibody binding to the NH(2)-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by the altered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to the NH(2)-terminal extension had effects on the interaction of TnT with troponin I, troponin C, and most significantly, tropomyosin. The data indicate that the NH(2)-terminal Tx [4-7 repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slow skeletal muscle TnT.
Jin
J P
JP
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4970, USA. jxj12@po.cwru.edu
Chen
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A
Ogut
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Huang
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism*; D018657:Muscle Fibers, Slow-Twitch / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000378:metabolism; D019209:Troponin C / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D015032:Zinc; D002118:Calcium