91 the structural changes occurring in cam as ca 2

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91 The structural changes occurring in CaM as Ca 2 + ions are bound are asso- ciated with pronounced changes in IH NMR, UV, fluorescence, and CD spec- tra. 87 The observed changes in CD and fluorescence spectra in the presence of Mg 2 + are only about 20 to 25 percent of those induced by Ca 2+ . A comparison of the CD spectra of CaM and its tryptic fragments indicates that the structural changes induced by Ca 2+ are substantially greater in the C-terminal than in the N-terminal half. 92 By and large, few structural details of the conformation changes have as yet been obtained. However, one aspect of the Ca 2 + -induced confor- mation change is that hydrophobic sites, probably one on each domain of the molecule, become exposed. In the presence of excess Ca 2 +, CaM will bind to other hydrophobic molecules, e.g., phenyl-Sepharose, a variety of drugs, many small peptides, and-last but not least-its target proteins. This brings us to the question of how CaM recognizes and interacts with the latter. We may suspect that the hydrophobic sites on each domain are somehow involved, but the role played by the central helix is still not clear. To explain small-angle x-ray scat- tering data, the interconnecting helix needs to be kinked, bringing the intact globular domains closer. 93 A putative CaM-binding segment (27 amino acids long) of myosin light- chain kinase (MLCK), an enzyme activated by CaM, has been identified. 94 The interaction between the segment peptide ("M13") and CaM has been studied 95 by CD spectroscopy and IH NMR. From these studies it appears that a unique 1: 1 complex is formed, and that secondary and tertiary structural changes occur not only in the peptide M13 but also in both halves of the CaM molecule. Further NMR studies 96,97 of the interaction between CaM and naturally occur- ring peptides (mellitin and mastoparan) that share some structural features of M13---clusters of basic residues, hydrophobic residues adjacent to the basic res- idues, and a predicted high a-helical content-show very much the same re- sults. Based on these results, a model, shown in Figure 3.18, for the interaction between CaM and M13 has been proposed. In this model the central helix is kinked at position 81, allowing the two domains to wrap around the assumed a-helical M13. Preliminary structure calculations of calcium-loaded CaM, based on NMR data, indicate that the central helix in solution indeed is kinked and very flexible,99 and comparisons 100 of chemical shifts in calmodulin with and without M13 complexed supports the model in Figure 3.18. Recent structural studies using NMR spectroscopy and x-ray diffraction have essentially con- firmed the general features of this model, although the orientation of the peptide is found to be reversed. 173 In conclusion, two important features of the protein should be recognized.
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