Cloning and expression of salmon cardiac troponin C: titration of the low-affinity Ca(2+)-binding site using a tryptophan mutant.
Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.
Moyes
C D
CD
Department of Biology, Queen's University, Kingston, Ontario, Canada. moyesc@biology.queensu.ca
Borgford
T
T
LeBlanc
L
L
Tibbits
G F
GF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D015870:Gene Expression; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009206:Myocardium / Q000737:chemistry*; D017686:Oncorhynchus mykiss; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012474:Salmon; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019209:Troponin C; D014364:Tryptophan
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014336:Troponin; D019209:Troponin C; D014364:Tryptophan; D002118:Calcium