Ca2+, Mg2+, and troponin I inhibitory peptide binding to a Phe-154 to Trp mutant of chicken skeletal muscle troponin C.
The effects of Ca2+, Mg2+, and troponin I (TnI) inhibitory peptide (Ip) binding on the spectral properties of a Phe-154 to Trp mutant (F154W) of chicken recombinant troponin C (rTnC) have been examined. Residue 154 is positioned in the final flanking helix H of metal binding site IV. Since there are no other Tyr or Trp residues in the protein, spectral properties can be unambiguously assigned. No significant differences in the far UV CD spectra of rTnC and F154W were observed in either the absence or presence of Ca2+. When reconstituted into whole Tn the ATPase specific activities (+/- Ca2+) of the troponin-tropomyosin-actomyosin subfragment 1 system were the same for both proteins. A 2-fold reduction in Ca2+ affinity of C domain sites III/IV but not of N domain sites I/II in isolated F154W is explicable in terms of the environment of residue 154 in the relatively disordered apo-C domain and its buried position in the known ordered 2Ca2+ crystal structure. Filling of sites III/IV by divalent cations was accompanied by a number of spectral changes which were different for Ca2+ and Mg2+. Binding of Ip peptides (residues 96-116 and 104-115(116)) elicited fluorescence emission spectral alterations in the presence of Ca2+. These were not observed in its absence nor in the presence of Mg2+ even though binding occurs under these conditions. Since Ca2+ affinity to C domain but not to N domain sites was increased by Ip at the low concentrations of protein and Ip tested, Ip binding appears to be stronger with C domain.(ABSTRACT TRUNCATED AT 250 WORDS)
Chandra
M
M
Department of Biochemistry, University of Alberta, Edmonton, Canada.
McCubbin
W D
WD
Oikawa
K
K
Kay
C M
CM
Smillie
L B
LB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D002942:Circular Dichroism; D017931:DNA Primers; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I; D014364:Tryptophan
D017931:DNA Primers; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D010649:Phenylalanine; D004533:Egtazic Acid; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium