Troponins

Proteins can be broadly classified into fibrous and globular. Many fibrous proteins serve a stmctural role (11). CC-Keratin has been described. Fibroin, the primary protein in silk, has -sheets packed one on top of another. CoUagen, found in connective tissue, has a triple-hehcal stmcture. Other fibrous proteins have a motile function. Skeletal muscle fibers are made up of thick filaments consisting of the protein myosin, and thin filaments consisting of actin, troponin, and tropomyosin. Muscle contraction is achieved when these filaments sHde past each other. Microtubules and flagellin are proteins responsible for the motion of ciUa and bacterial dageUa. 

Calcium is the trigger behind the muscle contraction process (24,25). Neural stimulation activates the release of stored Ca(Il) resulting in a dramatic increase in free calcium ion levels. The subsequent binding of Ca(Il) resulting in a dramatic increase in free calcium ion levels. The subsequent binding of Ca(Il) to the muscle protein troponin C provides the impetus for a conformational change in the troponin complex and sets off successive events resulting in muscle contraction. 

The first sequence is from the enzyme citrate synthase, residues 260-270, which form a buried helix the second sequence is from the enzyme alcohol dehydrogenase, residues 355-365, which form a partially exposed helix and the third sequence is from troponin-C, residues 87-97, which form a completely exposed helix. Charged residues are colored red, polar residues ate blue, and hydrophobic residues are green. 

Table 2.2 Amino acid sequences of calcium-binding EF motifs in three different proteins Pamalbumin VKKAFAI I DQDKSGFIEEDELKLFLQNF Calmodulin FKEAFSLFDKDGDGT I TTKELGTVMRSL Troponin-C LADCFR I FDKNADGF I D lEELGE I LRAT...

Herzberg, O., James, M.N.G. Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution. Nature 313 653-659, 1985. 

Figure 6.21a) comprising two domains separated by a long straight a helix, similar in shape to troponin-C described in Chapter 2 (see Figure 2.13c). Each domain comprises two EF hands (see Figure 2.13a), each of which binds a calcium atom. The two domains are clearly separated in space at the two ends of the a helix linker. 

Nonrepetitive but well-defined structures of this type form many important features of enzyme active sites. In some cases, a particular arrangement of coil structure providing a specific type of functional site recurs in several functionally related proteins. The peptide loop that binds iron-sulfur clusters in both ferredoxin and high potential iron protein is one example. Another is the central loop portion of the E—F hand structure that binds a calcium ion in several calcium-binding proteins, including calmodulin, carp parvalbumin, troponin C, and the intestinal calcium-binding protein. This loop, shown in Figure 6.26, connects two short a-helices. The calcium ion nestles into the pocket formed by this structure. 

In addition to the major proteins of striated muscle (myosin, actin, tropomyosin, and the troponins), numerous other proteins play important roles in the maintenance of muscle structure and the regulation of muscle contraction. Myosin and actin together account for 65% of the total muscle protein, and tropomyosin and the troponins each contribute an additional 5% (Table 17.1). The other regulatory and structural proteins thus comprise approximately 25% of the myofibrillar protein. The regulatory proteins can be classified as either myosin-associated proteins or actin-associated proteins. 

Troponin C Troponin I Troponin T Minor M protein 18 21 31 165 2 M line Ca binding Inhibits actin-myosin interaction Binds to tropomyosin Binds to myosin... 

Actin thin filaments consist of actin, tropomyosin, and the troponins in a 7 1 1 ratio (Figure 17.15). Each tropomyosin molecule spans seven actin molecules, lying along the thin filament groove, between pairs of actin monomers. 

FIGURE 17.29 A drawing of the thick and thin filaments of skeletal mnscle in cross-section showing the changes that are postulated to occur when Ca binds to troponin C. 

FIGURE 17.30 (a) A ribbon diagram and (b) a molecular graphic showing two slightly different views of the structure of troponin C. Note the long a-helical domain connecting the N-terminal and C-terminal lobes of the molecule. 

Farah, C., and Reinach, F., 1995. The troponin complex and regulation of muscle contraction. The FASEB Journal 9 755-767. 

The Ca2+-binding subunit TN-C is homologous to calmodulin with four EF-hands. In contrast to calmodulin, which is ubiquitously expressed in multicellular eukaryotic organisms and interacts with many targets, troponin specifically regulates muscle contraction. There are some structural differences between Troponin C in skeletal and cardiac muscles reflecting their physiological differences. 

Troponin is a regulator of striated muscle contraction. Measurements of troponin I levels are routinely used in the diagnosis of myocardial infarction. In addition, mutations in the troponin I subunit are associated with familial hypertrophic cardiomyopathy. 

Troponin specifically regulates muscle contraction. Ca2+-Binding Proteins... 

Muscle contraction is initiated by a signal from a motor nerve. This triggers an action potential, which is propagated along the muscle plasma membrane to the T-tubule system and the sarcotubular reticulum, where a sudden large electrically excited release of Ca " into the cytosol occurs. Accessory proteins closely associated with actin (troponins T, I, and C) together with tropomyosin mediate the Ca -dependent motor command within the sarcomere. Other accessory proteins (titin, nebulin, myomesin, etc.) serve to provide the myofibril with both stability... 

The regulation of smooth muscle and nonmuscle myosin-II is substantially different from the mechanism described above for two important reasons. First, there is no troponin in smooth muscle and nonmuscle cells. Second, although the rate of hydrolysis of ATP by these myosins is low in the presence of physiological concentrations of Mg % the addition of actin does not necessarily result in the stimulation of ATP hydrolysis by smooth muscle or nonmuscle myosin-II. These observations suggest the presence of a unique mechanism for Ca " regulation in smooth and nonmuscle cells, and that these myosins require an activation process before actin can stimulate ATP hydrolysis. 

Tropomyosin is thought to lie in the groove formed between the associated actin strands. The sites at which the myosin crossbridges attach are affected by the relationship between tropomyosin and the actin strands. The role of tropomyosin in smooth muscle is completely undefined while in striated muscle it is clearly involved in the activation of contraction. The difference is made clear by the absence from smooth muscle of the protein, troponin, which in striated muscle provides the binding site for the triggering calcium. 

The superstructure of smooth muscle actin filaments is differentiated from those of striated muscle by the absence of the troponins and the lateral organization by association of the filaments with dense bodies instead of with the Z-line. How these differences are encoded is again not at all clear. However, the myofibrillar structure and the alignment of the alternating actin and myosin filaments is apparently due primarily to dense bodies and the actin-actinin macrostructures. As the bent dumbbell shaped actins assemble into filaments they are all oriented in the same direction. The S-1 fragments of myosin will bind to actin filaments in vitro and in... 

Of the several kinase activities which are important in smooth muscle, myosin light chain kinase, MLCK, is the one responsible for activation of the actin-myosin system to in vivo levels. MLCK is present in the other nonmuscle cell types which have the actin-myosin contractile system and all of these are probably activated in a manner similar to smooth muscle rather than by way of the Ca -troponin mechanism of striated muscle. MLCK from smooth muscle is about 130 kDa and is rather variable in shape. It is present in smooth muscle in 1-4 pM concentrations and binds with an equally high affinity to both myosin and actin. Thus, most MLCK molecules are bound to actin. Myosin light chain serine-19 is the primary target of smooth muscle myosin light chain kinase. 

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