Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations.
To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.
Pearlstone
J R
JR
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Chandra
M
M
Sorenson
M M
MM
Smillie
L B
LB
D000409:Alanine / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D014466:Ultraviolet Rays
D011994:Recombinant Proteins; D019209:Troponin C; D018698:Glutamic Acid; D014364:Tryptophan; D000409:Alanine; D002118:Calcium