Abstract
To investigate how Ca2+ binding to troponin C regulates muscle contraction, the Ca(2+)-sensitive properties of thin filament assembly were studied as the tropomyosin binding, NH2-terminal region of troponin T was progressively shortened. Troponin complexes were prepared that contained skeletal muscle troponin C, troponin I, and either intact troponin T (TnT) (residues 1-259) or fragment TnT-(70-259), TnT-(151-259), or TnT-(159-259). In the absence of Ca2+ their respective affinities for pyrene-labeled tropomyosin were 2.3 x 10(7) M-1, 1.2 x 10(7) M-1, 1.9 x 10(5) M-1, and 1.9 x 10(5) M-1. Ca2+ had only a small effect on these affinities: 1.1 x 10(7) M-1 for whole troponin, 2 x 10(5) M-1 for troponin-(151-259), and 2.8 x 10(5) M-1 for troponin-(159-259). Forms of troponin that bound weakly to tropomyosin in the absence of actin increased the actin affinity of tropomyosin only 2-3-fold, even in the absence of Ca2+; weak binding of troponin to tropomyosin correlated with weak effects on tropomyosin-actin binding. In contrast, whole troponin had an approximately 500-fold effect on tropomyosin binding to actin, regardless of whether Ca2+ was present. The small effect of Ca2+ on the energetics of thin filament assembly is not attributable to the amino-terminal region of troponin T. The results suggest that Ca2+ causes the interaction between actin and the globular region of troponin to switch between two energetically similar states.
Highlights
Et al, 1983; Cho and Hitchcock-DeGregori, 1990; Ishii and Lehrer, 1991)
Under conditions of low tropomyosin concentration, which facilitates measurement of tight binding, the averages of several experiments as shown in Fig. 1 imply that removal of Ca2ϩ from troponin strengthens its affinity for tropomyosin about 2-fold, from 1.1 Ϯ 0.4 ϫ 107 MϪ1 in the presence of 50 mM CaCl2 to 2.3 Ϯ 0.3 ϫ 107 MϪ1 in the presence of EGTA (Table I). This small, Ca2ϩ-dependent change is consistent with similar results using cardiac troponin (Dahiya, et al, 1994), with a 1.7-fold increase in skeletal muscle troponin-tropomyosin photocross-linking in the absence of Ca2ϩ (Chong and Hodges, 1982) and is in the direction suggested by the standard model for troponin function, referenced earlier
2) The amino-terminal region of troponin T (TnT), especially residues 70 – 150, is required for tight association of troponin to tropomyosin. These results are consistent with observations involving TnT fragments, as opposed to the ternary troponin complexes in the present work
Summary
TnI, TnC, TnT, troponin I, C, and T, respectively; troponin-(159 –259), troponin produced by chymotryptic removal of TnT residues [1–158] from rabbit fast skeletal muscle whole troponin; troponin-(151–259), troponin reconstituted from recombinant rat fast skeletal muscle TnT fragment [151–259] and rabbit fast skeletal muscle TnC and TnI; troponin-(70 –259), reconstituted chimeric troponin, except the rat TnT fragment begins at Met-70. An alternative approach is to examine the properties of the Ca2ϩ-sensitive region of troponin (TnI, TnC, and the carboxyl terminus of TnT), in the absence of the apparently more Ca2ϩinsensitive tail region (i.e. the elongated amino-terminal portion of TnT). Because it lacks the Ca2ϩ-insensitive, anchoring portion of TnT, the effect of this truncated troponin complex on thin filament assembly may be very Ca2ϩ-sensitive. Binding of Truncated Troponin to Actin and Tropomyosin had a minimal effect on tropomyosin-actin binding, again regardless of whether Ca2ϩ was present These results have significance for how Ca2ϩ regulates muscle contraction and for the participation of different regions of troponin in thin filament assembly and regulation
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