160 the amino acid sequence of the yeast cam 147 aa m

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160 The amino-acid sequence of the yeast CaM (147 a.a.; M r = 16.1 kDa) is 60 percent identical with the sequences of all other CaMs known. In fact, if generally accepted conservative amino-acid replacements are allowed, the ho- mology increases to 80 percent or more, the most highly conserved portions being the four putative Ca 2 + -binding sites. Sites I and III match the EF-hand test sequence (see Figure 3.24) very well; in site a His occurs after the "z"- ligand instead of the archetypal Gly; and in site IV there is no amino acid between the residues that usually make up ligands "x" and "y." The effect of these alterations on the Ca 2+ affinity of yeast CaM is not yet known. That CaM is essential for the growth of yeast cells was shown by deletion or disruption of the gene. This constitutes, in fact, the first demonstration in any organism that CaM is an essential protein. (Deletions of genes in mammals are ethically questionable research procedures!) In the biochemically less sophiscated (than eukaryotes) prokaryotic cells, a regulatory role of Ca 2+ is not well-established. What is known is that calcium is massively accumulated during sporulation in many bacteria, for example, in strains of Bacillus, Streptomyces, and Myxococcus. In Myxococcus xanthus a development-specific protein called protein S assembles at the surface of myxos- pores in the presence of Ca 2 +. The DNA sequence of the gene that encodes this
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160 3 I CALCIUM IN BIOLOGICAL SYSTEMS protein has been deciphered. 161 The primary sequence of protein S (175 a.a., M r = 19.2 kDa) turns out to closely resemble mammalian CaM. It has four internally homologous regions with putative Ca 2+ sites. At least two of these are partly similar to the typical EF-hand, but uncharacteristically there are many more prolines in the M. xanthus protein than in bovine CaM (12 versus 2); so it is questionable if the bacterial protein really has the repeated helix-loop-helix structure found in mammalian CaM. 162 One candidate for a prokaryotic CaM was reported by Leadlay et al. 163 in Streptomyces erythreaus, the bacterium that produces the well-known antibiotic "erythromycin." The amino-acid sequence of a low-molecular-weight Ca 2 +- binding protein, as determined from the gene encoding it, revealed a high ho- mology with mammalian CaM. The protein is made up of 177 amino acids (M r = 20.1 kDa), and has four regions that are predicted to have the helix- loop-helix secondary structure typical of EF-hand proteins. The aligned se- quences of the 12 residues in each of the four potential calcium-binding loops in the S. erythreaus protein are compared with those of human calmodulin in Table 3.6. The pattern of residues in the S. erythraeus protein is typical of an EF-hand at least in sites I, III, and IV. Site II is unusual in having Gly at both positions I and 3. I13Cd NMR studies show that the bacterial protein binds three metal ions strongly (K :2: 105 M -1) with chemical shifts close to those expected for EF-hands, and lH NMR studies show that it undergoes a Ca 2 +- dependent conformational change.
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