Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

Abstract

The direct binding of Ca2+ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnCDANZ) was measured (25 degrees C) at three Mg2+ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca2+ to high- and low-affinity sites of troponin. The binding of Ca2+ to the high-affinity sites had a greater sensitivity to Mg2+ (KMg = 1 x 10(4)M-1) than the low-affinity sites (KMg = 1.2 x 10(3)M-1). The fluorescence change of thin filaments reconstituted with TnCDANZ was titrated with Ca2+ in the same solutions used for binding assays. The Ca2+-dependent fluorescence change had nearly the same sensitivity to Mg2+ (KMg = 9.4 x 10(2)M-1) as did Ca2+ binding to the low-affinity sites. The Ca2+ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca2+ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca2+ binding to the low-affinity sites of isolated TnCDANZ was measured (4 degrees C). From these results the binding of Ca2+ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca2+ affinity.

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

“…With troponin, for example, the concentration of Ca-regulatory sites is ~240 I~M (Baylor et al, 1983) and their KD is expected to be ~<2 I~M (Zot and Potter, 1987 …”

Switch Action of Troponin on Muscle Thin Filament as Revealed by Spin Labeling and Pulsed EPR

“…3b, left panel). However, a small difference in the distances may explain why the Ca 2ϩ affinity is severalfold weaker for the filament than for the complex alone (46,47). Our previous spin-labeling study showed that the geometry of TnC differed slightly (by Ͻ1.5 Å) in the monomer and in the TnC-I complex, to explain their nearly 10-fold affinity difference (10).…”

Engineering Competitive Magnesium Binding into the First EF-hand of Skeletal Troponin C

“…3 (15,19,23,27,32). Several groups demonstrated that as the [Mg 2ϩ ] was increased, the Ca 2ϩ sensitivity of TnC, Tn-activated actomyosin ATPase, and force development decreased (19,(23)(24)(25)(26)(27)(28)(29)(30)(31) 50 caused by incomplete force recovery was calculated for each mutant using the linear fit to the partial TnC extraction data in Fig. 7C (- 50 , which was then converted to pCa 50 . was similar to that of psoas muscle fibers reconstituted with cTnC (69,70).…”

“…21 and 22). However, addition of Mg 2ϩ has been shown to decrease the Ca 2ϩ sensitivity of the regulatory domain of fluorescent TnCs in isolation (19,(23)(24)(25)(26)(27), in the Tn complex (23,28), and in reconstituted muscle fibers (26,29). Furthermore, several groups have dem- 1 The abbreviations used are: TnC, intact chicken skeletal troponin C; Tn, troponin; TnC F29W , intact TnC mutant with the Phe 29 3 Trp mutation; G34DTnC F29W , intact TnC F29W mutant with the Gly 34 3 Asp mutation; TnC 1-90 F29W , isolated N-terminal domain representing residues 1-90 of TnC F29W ; cTnC F27W , intact human cardiac TnC mutant with the Phe 27 3 Trp mutation; TnI, chicken skeletal troponin I; TnI-(96 -148), chicken skeletal troponin I peptide corresponding to residues 96 -148; CaM, calmodulin; CaM F19W , CaM mutant with the Phe 19 3 Trp mutation; pCa, -log[Ca 2ϩ ]; [Ca 2ϩ ] 50 , [Ca 2ϩ ] that produced half-maximal force; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; Quin-2, 2-((bis(carboxymethyl)amino)-5-methylphenoxy)-methyl)-6-methoxy-8-(bis(carboxymethyl)amino)-quinoline.…”