Modulation of troponin T molecular conformation and flexibility by metal ion binding to the NH2-terminal variable region.
Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca(2+)-regulation of striated muscle contraction. Developmentally regulated alternative RNA splicing produces TnT isoforms differing in their NH(2)-terminal structure. Physical property variations of the NH(2)-terminal hypervariable region of TnT may have a role in tuning the Ca(2+)-sensitivity and overall cooperativity of the muscle. We have previously demonstrated that metal ion or monoclonal antibody binding to the NH(2)-terminal region can modulate the epitopic conformation and troponin I and tropomyosin binding affinity of TnT. To further establish the molecular basis of this conformational and functional modulation, we have characterized the NH(2)-terminal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isoform, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx) in the NH(2)-terminal variable region was used in this study. TnT8e16 was titrated for Cu(II) binding-induced changes in fluorescence intensity and anisotropy of the COOH-domain Trp residues (W234, W236, and W285), which demonstrated considerable environmental sensitivity in TnT denaturation studies. Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion binding-induced conformational change in TnT. Fluorescence anisotropy changes upon metal ion binding indicated a decrease in the mobility of the Trp residues and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH(2)-terminal variable region of TnT modulates conformation and flexibility of other domains of the protein.
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Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4970, USA. jxj12@po.cwru.edu
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D017398:Alternative Splicing; D000818:Animals; D000906:Antibodies / Q000032:analysis; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D018604:Epitope Mapping; D005454:Fluorescence Polarization; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D020107:Troponin T / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D000906:Antibodies; D002413:Cations, Divalent; D010446:Peptide Fragments; D020033:Protein Isoforms; D012996:Solutions; D020107:Troponin T; D003300:Copper; D014364:Tryptophan; D015032:Zinc