Calmodulins

Barbate G, Ikura M, Kay L E, Pastor R W and Bax A 1992 Backbone dynamics of calmodulin studied by N relaxation using inverse detected two-dimensional NMR spectroscopy the central helix is flexible S/oefrem/sf/ y 31 5269-78... 

Although intended for the biochemistry lab, this experiment provides analytical students with a practical characterization analysis. Of particular interest is the use of Job s method to determine the number of TNS (2-p-toludinylnaphthalene-6-sulfonate) binding sites on calmodulin, fluorescence is measured at 475 nm using an excitation wavelength of 330 nm. 

In addition, vinpocetine selectively inhibits a specific calcium, calmodulin-dependent cycHc nucleotide phosphodiesterase (PDF) isozyme (16). As a result of this inhibition, cycHc guanosine 5 -monophosphate (GMP) levels increase. Relaxation of smooth muscle seems to be dependent on the activation of cychc GMP-dependent protein kinase (17), thus this property may account for the vasodilator activity of vinpocetine. A review of the pharmacology of vinpocetine is available (18). 

Gofactors. Frequendy proteins exist in their native state in association with other nonprotein molecules or cofactors, which are cmcial to their function. These may be simple metal ions, such as Fe " in hemerythrin or Ca " in calmodulin a heme group, as for the globins nucleotides, as for dehydrogenases, etc. 

Another mechanism in initiating the contraction is agonist-induced contraction. It results from the hydrolysis of membrane phosphatidylinositol and the formation of inositol triphosphate (IP3)- IP3 in turn triggers the release of intracellular calcium from the sarcoplasmic reticulum and the influx of more extracellular calcium. The third mechanism in triggering the smooth muscle contraction is the increase of calcium influx through the receptor-operated channels. The increased cytosolic calcium enhances the binding to the protein, calmodulin [73298-54-1]. 

Diltiazem inhibits calcium influx via voltage-operated channels and therefore decreases intracellular calcium ion. This decreases smooth muscle tone. Diltiazem dilates both large and small arteries and also inhibits a-adrenoceptor activated calcium influx. It differs from verapamil and nifedipine by its use dependence. In order for the blockade to occur, the channels must be in the activated state. Diltiazem has no significant affinity for calmodulin. The side effects are headache, edema, and dizziness. 

Heavy metal contamination of pH buffers can be removed by passage of the solutions through a Chelex X-100 column. For example when a solution of 0.02M HEPES [4-(2-HydroxyEthyl)Piperazine-l-Ethanesulfonic acid] containing 0.2M KCl (IL, pH 7.5) alone or with calmodulin, is passed through a column of Chelex X-100 (60g) in the K" " form, the level of Ca ions falls to less than 2 x 10" M as shown by atomic absorption spectroscopy. Such solutions should be stored in polyethylene containers that have been washed with boiling deionised water (5min) and rinsed several times with deionised water. TES [, N,N, -Tetraethylsulfamide] and TRIS [Tris-(hydroxymethyl)aminomethane] have been similarly decontaminated from metal ions. 

Recrystd from abs EtOH dried in vacuo and stored in tightly stoppered bottles because it is hygroscopic. It is soluble in H2O but insoluble in CgHg, Et20 and alkaline aqueous soln. It has UV at 258 and 307.5nm (log e 4.50 and 3.50) in EtOH (neutral species). [Craig et al. J Org Chem 22 709 1957.] It is a calmodulin inhibitor [Levene and Weiss J Parmacol Exptl Ther2Q% 454 1978], and js a psychotropic agent [Fowler Arzneim.-Forsch 27 866 1977]. 

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...

Peptide binding to calmodulin induces a large interdomain movement... 

The x-ray structure of free calmodulin was determined by the group of Charles Bugg, University of Alabama. It is a dumbbell-shaped molecule... 

Figure 6.21 Schematic diagram of the conformational changes of calmodulin upon peptide binding, (a) In the free form the calmodulin molecule is dumhhell-shaped comprising two domains (red and green), each having two EF hands with bound calcium (yellow), (b) In the form with bound peptides (blue) the a helix linker has been broken, the two ends of the molecule are close together and they form a compact globular complex. The internal structure of each domain is essentially unchanged. The hound peptide binds as an a helix.

Finn, B.E., Forsen, S. The evolving model of calmodulin structure, function and activation. Structure 3 7-11, 1995. 

Babu, Y.S., et al. Three-dimensional structure of calmodulin. Nature 315 37-40, 1985. 

Ikura, M., et al. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256 632-638, 1992. 

Meador, W.E., Means, A.R., Quiocho, F.A. Target enzyme recognition by calmodulin 2.4 A stmcture of a calmodulin-peptide complex. Science 257 1251-1255, 1992. 

FIGURE 6.24 (a) The alpha helix consisting of residues 153-166 (red) in flavodoxin from Anahaena is a surface helix and is amphipathic. (b) The two helices (yellow and blue) in the interior of the citrate synthase dimer (residues 260-270 in each monomer) are mostly hydrophobic, (c) The exposed helix (residues 74-87—red) of calmodulin is entirely accessible to solvent and consists mainly of polar and charged residues. 

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. 

Smooth muscle contractions are subject to the actions of hormones and related agents. As shown in Figure 17.32, binding of the hormone epinephrine to smooth muscle receptors activates an intracellular adenylyl cyclase reaction that produces cyclic AMP (cAMP). The cAMP serves to activate a protein kinase that phosphorylates the myosin light chain kinase. The phosphorylated MLCK has a lower affinity for the Ca -calmodulin complex and thus is physiologically inactive. Reversal of this inactivation occurs via myosin light chain kinase phosphatase. 

There are numerous second messenger systems such as those utilizing cyclic AMP and cyclic GMP, calcium and calmodulin, phosphoinosiddes, and diacylglerol with accompanying modulatory mechanisms. Each receptor is coupled to these in a variety of ways in different cell types. Therefore, it can be seen that it is impractical to attempt to quantitatively define each stimulus-response mechanism for each receptor system. Fortunately, this is not an... 

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