Characterization of the biologically important interaction between troponin C and the N-terminal region of troponin I.
The N-terminal regulatory region of Troponin I, residues 1-40 (TnI 1-40, regulatory peptide) has been shown to have a biologically important function in the interactions of troponin I and troponin C. Truncated analogs corresponding to shorter versions of the N-terminal region (1-30, 1-28, 1-26) were synthesized by solid-phase methodology. Our results indicate that residues 1-30 of TnI comprises the minimum sequence to retain full biological activity as measured in the acto-S1-TM ATPase assay. Binding of the TnI N-terminal regulatory peptides (TnI 1-30 and the N-terminal regulatory peptide (residues 1-40) labeled with the photoprobe benzoylbenzoyl group, BBRp) were studied by gel electrophoresis and photochemical cross-linking experiments under various conditions. Fluorescence titrations of TnI 1-30 were carried out with TnC mutants that carry a single tryptophan fluorescence probe in either the N- or C-domain (F105W, F105W/C domain (88-162), F29W and F29W/N domain (1-90)) (Fig. 1). Low Kd values (Kd < 10(-7) M) were obtained for the interaction of F105W and F105W/C domain (88-162) with TnI 1-30. However, there was no observable change in fluorescence when the fluorescence probe was located at the N-domain of the TnC mutant (F29W and F29W/N domain (1-90)). These results show that the regulatory peptide binds strongly to the C-terminal domain of TnC.
Ngai
S M
SM
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. smngai@cuhk.edu.hk
Pearlstone
J R
JR
Smillie
L B
LB
Hodges
R S
RS
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D009154:Mutation; D009206:Myocardium; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D000199:Actins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014335:Tropomyosin; D019209:Troponin C; D019210:Troponin I; D014364:Tryptophan; D009218:Myosins; D002118:Calcium