title: Calcium-regulated conformational change in the C-terminal end segment of troponin I and its binding to tropomyosin.
abstract: The troponin complex plays an essential role in the thin filament regulation of striated muscle contraction. Of the three subunits of troponin, troponin I (TnI) is the actomyosin ATPase inhibitory subunit and its effect is released upon Ca(2+) binding to troponin C. The exon-8-encoded C-terminal end segment represented by the last 24 amino acids of cardiac TnI is highly conserved and is critical to the inhibitory function of troponin. Here, we investigated the function and calcium regulation of the C-terminal end segment of TnI. A TnI model molecule was labeled with Alexa Fluor 532 at a Cys engineered at the C-terminal end and used to reconstitute the tertiary troponin complex. A Ca(2+) -regulated conformational change in the C-terminus of TnI was shown by a sigmoid-shape fluorescence intensity titration curve similar to that of the CD calcium titration curve of troponin C. Such corresponding Ca(2+) responses are consistent with the function of troponin as a coordinated molecular switch. Reconstituted troponin complex containing a mini-troponin T lacking its two tropomyosin-binding sites showed a saturable binding to tropomyosin at pCa 9 but not at pCa 4. This Ca(2+) -regulated binding was diminished when the C-terminal 19 amino acids of cardiac TnI were removed. These results provided novel evidence for suggesting that the C-terminal end segment of TnI participates in the Ca(2+) regulation of muscle thin filament through interaction with tropomyosin.
authors.affiliation: Evanston Northwestern Healthcare and Northwestern University, Evanston, IL, USA.
authors.forename: Zhiling
authors.initials: Z
authors.lastname: Zhang
authors.forename: Shirin
authors.initials: S
authors.lastname: Akhter
authors.forename: Steven
authors.initials: S
authors.lastname: Mottl
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Jin
mesh_terms: D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007211:Indoles / Q000737:chemistry; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*; D014336:Troponin / Q000737:chemistry; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020107:Troponin T / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014966:Xanthenes / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D019210:Troponin I; D020107:Troponin T; D014966:Xanthenes; C042260:troponin-tropomyosin complex; D002118:Calcium