Calmodulin, calcium binding

Once the intracellular Ca " concentration begins to rise, calmodulin-calcium binding also rises and MLCK, which is dependent on calmodulin activation, rises in turn. The next step in this cascade is the phosphorylation of myosin. Finally, the phosphorylation of myosin results in the activation of the crossbridges and the accompanying transduction of ATP energy into mechanical work. Despite its differences in regulation, smooth muscle behaves mechanically much like other muscles. 

M.-C. Kilhoffer, J. G. Demaille, and D. Gerard, Terbium as luminescent probe of calmodulin calcium-binding sites. Domains I and II contain the high-affinity sites, FEBS Lett. 116, 269-272 (1980). 

Recently, a chameleon calcium reporter has been designed by R.Y. Tsien to sense calcium in cells by FRET. The structure of the system is based on a CEP separated from an YEP by the calmodulin calcium binding protein (CaM) and a calmodulin binding peptide (M13). If Ca ions are bound, CaM wraps around Ml 3 allowing high efficiency of excitation transfer from the donor CEP to the acceptor YEP (see Figure 9). The degree of FRET in chameleon is a sensitive ratiometric reporter (ratio 527/434 YPE/ CEP) of the calcium concentration in cells and in solution. The beauty of this approach is that the... 

One of these motifs, called the helix-turn-helix motif, is specific for DNA binding and is described in detail in Chapters 8 and 9. The second motif is specific for calcium binding and is present in parvalbumin, calmodulin, tro-ponin-C, and other proteins that bind calcium and thereby regulate cellular activities. This calcium-binding motif was first found in 1973 by Robert Kretsinger, University of Virginia, when he determined the structure of parvalbumin to 1.8 A resolution. 

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...

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. 

Small ubiquitous calcium-binding protein. Calmodulin binds and regulates the activity of many protein targets involved in cellular signal transduction pathways mediated by calcium. Calmodulin is ranked among the most conserved proteins and plays a key role in many cellular processes. 

Myosin-I molecules have several IQ sequences on or near the head and have light chains associated with them (Cheney and Mooseker, 1992 Cheney et al., 1993). Frequently, the light chains appear to be calmodulin molecules and some myosin-I molecules can bind three to four molecules of calmodulin at one time. Brush-border and adrenal myosin-I also bind calmodulin. Acanthamoeba myosin-I has a light chain that can be removed, in vitro, without adversely affecting the ATPase activity or the heavy chain phosphorylation (Korn and Hammer, 1988). The role of these calmodulin molecules in regulating myosin-I is complex and poorly understood. One possibility is that the calmodulin molecules dissociate from the heavy chains when calcium binds to the calmodulin, thereby imparting greater flexibility to the head of the myosin-I molecules. 

In striated muscle, there are two other proteins that are minor in terms of their mass but important in terms of their function. Tropomyosin is a fibrous molecule that consists of two chains, alpha and beta, that attach to F-actin in the groove between its filaments (Figure 49-3). Tropomyosin is present in all muscular and muscle-fike structures. The troponin complex is unique to striated muscle and consists of three polypeptides. Troponin T (TpT) binds to tropomyosin as well as to the other two troponin components. Troponin I (Tpl) inhibits the F-actin-myosin interaction and also binds to the other components of troponin. Troponin C (TpC) is a calcium-binding polypeptide that is structurally and functionally analogous to calmodulin, an important calcium-binding protein widely distributed in nature. Four molecules of calcium ion are bound per molecule of troponin C or calmodulin, and both molecules have a molecular mass of 17 kDa. 

Site of calcium binding Troponin Calmodulin Calmodulin... 

Fig. 5 Proposed signal transduction mechanisms that stimulate the pheromone biosynthetic pathway in Helicoverpa zea and Bombyx mori. It is proposed that PBAN binds to a G protein-coupled receptor present in the cell membrane that upon PBAN binding will induce a receptor-activated calcium channel to open causing an influx of extracellular calcium. This calcium binds to calmodulin and in the case of B. mori will directly stimulate a phosphatase that will dephosphorylate and activate a reductase in the biosynthetic pathway. In H. zea the calcium-calmodulin will activate adenylate cyclase to produce cAMP that will then act through kinases and/or phosphatases to stimulate acetyl-CoA carboxylase in the biosynthetic pathway...

Volume 102. Hormone Action (Part G Calmodulin and Calcium-Binding Proteins)... 

Calcium can also directly affect signaling through another pathway. The major calcium binding protein in the cell is calmodulin (CAM). CAM is not an enzyme, but it will activate some enzymes when it binds to them. CAM binds to its target enzymes only when calcium is bound... 

Table XI (346-390) lists a number of calcium-binding proteins and indicates very succinctly their role in biological systems. This table both illustrates the range of functions of calcium-binding proteins and serves to introduce those which appear subsequently in this chapter. The structures and functions of particularly important calcium-binding proteins such as calmodulin, parvalbumin, and troponin C are described in standard texts on biochemistry. The minimal Table XI entry for the particularly important calmodulins is amplified in the next paragraph. Table XI provides a sprinkling of references to enable readers to gain entry into the literature, for these and for most of the less-familiar species.

It has been shown that the activity of NO synthases is regulated by cofactors calcium binding protein calmodulin and tetrahydrobiopterin (H4B). Abu-Soud et al. [149] have studied the effect of H4B on the activity of neuronal nNOS I, using the isolated heme-containing oxygenase domain nNOSoxy. It was found that nNOSoxy rapidly formed an oxygenated complex in the reaction with dioxygen, which dissociated to produce superoxide (Reaction (6)) ... 

Calmodulin, a calcium binding protein, is involved in Ca2+-dependent regulation of several synaptic functions of the brain synthesis, uptake and release of neurotransmitters, protein phosphorylation and Ca+2 transport. It reacts with TET, TMT and TBT which then inactivates enzymes like Ca+2-ATPase and phosphodiesterase. In vitro studies indicated TBT was greater at inhibiting calmodulin activity than TET and TMT, whereas in vivo the order was TET > TMT > TBT. This may be due to the greater detoxification of TBT (66%) in the liver before moving to other organs30,31. 

A similar approach for detecting the presence of specific proteins has also been reported by Nilsson and coworkers [26]. In this study, a complex between a calmodulin, a small calcium-binding protein, and the zwitter-ionic polythiophene POWT was used to detect the presence of calcineurin. The interaction between the POWT-camodulin complex and calcineurin changed the emission profile from POWT, and no observable changes were observed upon exposure of the complex human serum albumin, suggesting that the complex could be used for the specific detection of calcineurin. 

The calcium mediated contraction of smooth muscle, which unlike striated muscle does not contain troponin, is quite different and requires a particular calcium-binding protein called calmodulin. Calmodulin (CM) is a widely distributed regulatory protein able to bind, with high affinity, four Ca2+ per protein molecule. The calcium—calmodulin (CaCM) complex associates with, and activates, regulatory proteins, usually enzymes, in many different cell types in smooth muscle the target regulatory proteins are caldesmon (CDM) and the enzyme myosin light chain kinase (MLCK). As described below, CaCM impacts on both actin and myosin filaments. 

The way in which calcium binding changes the tyrosine fluorescence of calmodulin was initially a controversial issue. In 1980, Seamon(n5) examined the binding of Ca2+ and Mg2+ by NMR and concluded that both Tyr-99 and Tyr-138 were perturbed by the first two calcium ions. Although Tyr-138 was also perturbed by the binding of the fourth calcium, both residues appeared to be associated with high-affinity domains (III and IV). 

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