Ebashi discovered troponin (TN) in 1963. Subsequently, Greaser and Gergely (1971) showed that troponin consists of three components. These protein subunits differ in their molecular weights (based on early electrophoretic studies), and each possesses a specific function (Fig. TN1).

Fig. TN1. SDS-PAGE of troponin.

Figure TN1a visualizes the three TN subunits interacting with tropomyosin and actin in the thin filament .

Fig. TN1a. A model of the molecular arrangement of TN subunits, TM, and actin in skeletal muscle thin filament (From Gordon et al., 2000). TN-I green, TN-C red, TN-T yellow. TM a-subunit brown, p-subunit orange. Actin gray. Note the adjacent TM molecules overlap head to tail with the N-terminus of TN-T lying along the overlap region.The C-terminus of TN-T interacts with TN-C and TNI, and TN-I also interacts with actin.

Figure TN1b shows the detailed interactions of the thin filament proteins pictured in Figure TN1a.

Fig. TN1b. Diagram indicating the effect of Ca-binding to TN-C on the interactions of the proteins shown in Fig. TN1a. (From Gordon et al. 2000). A, actin; I, TN-I; C, TN-C; T1, N-terminus of TN-T; T2, C-terminus of TN-T; Tm, tropomyosin with the N- and C- terminals indicated in the Head-to-tail overlap. Thicker lines imply stronger binding and weaker lines imply weaker binding. Left without bound Ca2+ , right with bound Ca. Note, Ca2+ -binding to TN-C enhances the TN-C--TN-I and TN-C--TN-T2 interactions and weakens the TN-I--A interactions.

Troponin C is the Ca2+ receptor in the thin filament. It has been crystallized and its three-dimensional structure determined (Herzberg and James, 1985; Houdusse et al., 1997). Fig. TN2. illustrates the structure.

Fig. TN2. Ribbon diagram of skeletal muscle TN-C. with one molecule of Ca2+ bound (left) and two molecules of Ca2+ bound (right). (From Grodon et al., 2000). Solid circles represent Ca2+, the letters A-H correspond to the helices, N is the amino terminal end and C is the carboxyl terminal end.

TN-C belongs to the Ca2+-binding protein family. Its Ca2+-binding domains comprise a short a-helix, a Ca2+-binding loop and a second short a-helix. The domain folds around the Ca2+ ion in a manner of a clenched right hand, with the extended forefinger and thumb representing the helices and the middle finger forming the loop structure. This domain is termed an E-F hand. Two E -F hand domains form a stable pair in many Ca2+-binding proteins. In TN-C, the two E-F hand domains are connected by a long a-helix resulting in four Ca2+-binding sites per molecule. Two of the four sites have high affinity to Ca2+ and are located in the carboxyl-terminal domain, whereas the other two sites have low affinity to Ca2+ and are located in the amino-terminal domain. Only the low affinity Ca2+-binding sites of TN-C are involved in the regulation of muscle contraction.

TN-C forms specific complexes with both TN-I and TN-T. The crystal structure of TN-C complexed with the N-terminal fragment of TN-I has been determined (Vassylyev et al., 1998). In the complex TN-C has a compact globular shape, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TN-C. The TN-I fragment binds to the N-terminal domain of TN-C in its Ca2+-bound conformation. Apparently, the complex formation between the two proteins requires a specific sterical configuration and, therefore, the complex has a functional significance.

2+ 2+ Troponin I inhibits the Mg2 -activated ATPase of actomyosin (identical with the actin and Mg2 -

activated myosin ATPase). TN-I is a basic protein that readily complexes with the acidic TN-C;

importantly Ca2+ strengthens the complex formation. Thus, upon muscle stimulation TN-C binds

Ca2+ and then complexes with TN-I; this provides a simple mechanism to relieve the inhibition of the actomyosin Mg2+-ATPase by TN-I. Studies with deletion mutants suggest that the C-terminal residues of TN-I are involved in the Ca2+ -sensitive molecular switch during muscle contration

(Ramos, 1999; Szczesna et al., 1999) Several isoforms of TN-I are known in the molecular mass range of 21-23 kDa.

Troponin T is an asymmetric molecule that interacts with TM. Evidence obtained from cocrystals of TN-T with TM suggests that TN-T is an asymmetric molecule that contacts the C-terminal region of TM along much of its length (Perry, 2001). Skeletal TN-T extends 160 A along the TM molecule whereas the cardiac isoform with a slightly longer polypeptide chain occupies 180 A.

The TM - TN-T interaction serves to fix the position of the entire TN complex within the thin filament, so that subtle changes in the conformation of these proteins may regulate contraction. In this respect, it is important that TN-T also binds TN-C, thus the Ca2+-induced conformational changes in TN-C are transmitted through TN-T to TM. A highly conserved protein domain was described in the amino acid sequence of TN-T (Stefancsik et al., 1998), that is characterized by a heptad repeat motif with a potential for a -helical coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TN-I sequences, suggesting that these protein domains play a role in TN-T - TN-I interaction. This is another example of how specific interactions of the TN subunits build the entire TN molecule.

TroponinT, similarly to TN-C and TN-I, has several isoforms in the molecular mass range of 30-35 kDa. Variations in Tn isoforms may modulate muscle performance.

The book of Perry (1996) is a good source for learning about the structure and properties of the troponin-tropomyosin system. Perry (1998 and 1999) also authored specific reviews on TN-T and TN-I. Furthermore, the review of Gordon et al. (2000) on the Regulation of Contraction in Striated Muscle includes a detailed discussion on the regulatory proteins.


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