Site specific mutagenesis

Tsuji, F. I., Inouye, S., Goto, T., and Sakaki, Y. (1986). Site-specific mutagenesis of the calcium-binding photoprotein aequorin. Proc. Natl. Acad. Sci. USA 83 8107-8111. 

Clusters Fa and Fb of photosystem I from cyanobacteria and chloro-plasts are distinguished by their EPR signatures (26, 27) and their reduction potentials (-520 mV for Fa and -580 mV for Fb Ref. (28). The assignment of cysteines in the primary sequence as ligands to individual clusters has been achieved by site-specific mutagenesis (29, Fig. 3), and structural information with regard to the environment of both clusters has been obtained by NMR (24). 

In the Ca-ATPase from sarcoplasmic reticulum, oligonucleotide-directed, site-specific mutagenesis has been applied to identify amino acids involved in Ca binding. Mutation of 30 glutamate and aspartate residues, singly or in groups, in a stalk sector near the transmembrane domain has little effect on Ca " -transport. In contrast mutations to Glu ° , Glu, Asn , Thr , Asp ° or Glu ° resulted in loss... 

Site specific mutagenesis of sacroplasmic reticulum -ATPase... 

The antiparallel strand structure between residues 131 and 238 in the cytoplasmic portion of Ca -ATPase was originally designated as transduction domain the name suggested its possible role in the conformational coupling between the nucleotide binding and phosphorylation sites exposed to the cytoplasm and the Ca channel located at some distance from each other in the lipid bilayer [8,42]. The site specific mutagenesis of conserved amino acids in the P strand sector of the molecule provides support for its proposed function in conformational transitions [103,126,127,215]. 

There is good agreement that the two high-affinity Ca sites are within = 10 A of each other (Table II) [132,390,404-409], Their localization within the bilayer is supported by the observation [130,131] that site-specific mutagenesis of several amino acids within the putative transmembrane helices interferes with Ca binding and with the Ca -dependent phosphorylation of the enzyme by ATP, but has no effect on the Ca -independent phosphorylation by inorganic phosphate. 

Site specific mutagenesis of sarcoplasmic reticulum Ca -ATPase. 

Booher, R., and Beach, D. (1986). Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccaromyces pombe. Mol. Cell. Biol. 6 3523-3530. Booher, R. N., Alfa, C. E., Hyams, J. S., and Beach, D. H. (1989). The fission yeast cdc2/cdcl3/sucl protein kinase regulation of catalytic activity and nuclear localization. CeU 58 485-497. 

Korotchkina, L.G. and Patel, M.S. (1995) Mutagenesis studies of the phosphorylation sites of recombinant human pyruvate dehydrogenase. Site-specific regulation. Journal of Biological Chemistry 270, 14297—14304. 

Aleshin and coworkers (49) have reported the X-ray crystal structure at 2.2-A resolution of a G2-type variant produced by Aspergillus awamori. Meanwhile, an attempt was made to determine the amino acid residues that participate in the substrate binding and catalysis provided by G2 of A. niger (52). The results of the chemical approach indicated that the Asp-176, Glu-179, and Glu-180 form an acidic cluster crucial to the functioning of the enzyme. This conclusion was then tested by site-specific mutagenesis of these amino acid residues, which were replaced, one at a time, with Asn, Gin, and Gin, respectively (53). The substitution at Glu-179 provided an inactive protein. The other two substitutions affected the kinetic parameters but were not of crucial importance to the maintenance of activity. The crystal structure (49) supports the conclusion that Glu-179 functions as the catalytic acid but Asp-17 6 does not appear to be a good candidate for provision of catalytic base. Thus, there still exists considerable uncertainty as to how the disaccharide is accepted into the combining site for hydrolysis. Nevertheless, the kind of scheme presented by Svensson and coworkers (52) almost surely prevails. 

---