The calcium‐induced switch in the troponin complex probed by fluorescent mutants of troponin I

• Published in: European journal of biochemistry Vol. 270; no. 14; pp. 2937 - 2944
• Main Authors: Oliveira, Deodoro C. S. G., Reinach, Fernando C.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.07.2003
• Subjects: 5-Hydroxytryptophan - chemistry; 5-Hydroxytryptophan - metabolism; 5‐hydroxytryptophan; Allosteric Regulation; Binding Sites; Ca2+‐binding protein; Calcium - chemistry; Calcium - metabolism; Calcium - pharmacology; Escherichia coli - genetics; Escherichia coli - metabolism; fluorescence; Mutagenesis, Site-Directed; Point Mutation - physiology; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; skeletal muscle; Spectrometry, Fluorescence; Tropomyosin - metabolism; Tropomyosin - physiology; troponin; Troponin - metabolism; Troponin - physiology; Troponin I - genetics; Troponin I - metabolism; Troponin I - physiology
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.1432-1033.2003.03659.x

The Ca2+‐induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5‐hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N‐domain of TnC are the inhibitory region (residues 96–116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N‐terminal domain of TnC might be the high affinity Ca2+‐binding sites in the troponin complex.