Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker.
Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.
Kozaili
Julie Mouannes
JM
Department of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Leek
Daniel
D
Tobacman
Larry S
LS
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D004533:Egtazic Acid / Q000737:chemistry; D006801:Humans; D054792:Muscle, Striated / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D015879:Myosin Subfragments; D010455:Peptides; D014335:Tropomyosin; D019210:Troponin I; D004533:Egtazic Acid; D002118:Calcium