Residuals deleted

Figure 38-5. Examples of the effects of deletions and insertions in a gene on the sequence of the mRNA transcript and of the polypeptide chain translated therefrom. The arrows indicate the sites of deletions or insertions, and the numbers in the ovals indicate the number of nucleotide residues deleted or inserted. Blue type indicates amino acids in correct order.

Fig. 6. Effects of C-terminal deletion of CM-ACSI on enzyme activity. A series of 3 -deletion mutants were prepared from cDNA for the wound-induced ACC synthase from winter squash as shown in solid bars. Numbers of the deletion mutants are same to the number of amino acid residues deleted from C-terminus. Activities of enzymes produced by the transformed E. coli were assayed and amounts of enzyme protein were estimated by ELISA. Relative specific activities of each mutant proteins are plotted against the nucleotide sequence. Amino acid sequence at the critical junction is also shown.

The X-SODb has the same overall tertiary and quaternary structure of all Cu2Zn2SODs reviewed so far, resulting in quite low values of 0.726 and 1.170 A for the RMS deviations of the Ca atoms in the bovine and yeast enz3mies, respectively. The SOD fold shows once again its robustness. Indeed, the several amino acid substitutions and the insertion in the bovine enzyme are absorbed well by the structure, the only substantially different part of it being the turn between residues 89 and 92, where a one-residue deletion occurs with respect to the bovine enzyme. It is of particular interest that the 10 substitutions of Pro and Gly residues in X-SODb and bovine SOD do not result in any evident changes in backbone conformations. 

The purification of the protected peptide intermediates is an important aspect of the CSPPS strategy to ensure homogeneous molecular species, free from single-residue deletion peptides and other impurities. However, a prerequisite for any chromatographic purification is adequate solubility of the material to be purified in a solvent compatible with the procedure. Protected peptides exhibit unpredictable, but generally poor, solubility in water and in most of the commonly used organic solvents, which makes them difficult to purify and causes some of the most serious problems in CSPPS. 

Point Mutations. Since the advent of recombinant DNA technology, a number of researchers have used point mutation techniques either to delete one or more residues within the hGH molecule or systematically to change from one amino acid to another to probe hGH stmcture/function relationships (33). 

A unique feature of the interaction of the hormone and PLR is at the beginning of the F-G loop in the C-terminal domain. In HGR the sequence is Arg-Asn-Ser whereas in PLR it is Asp-His-deletion. This loop interacts with His 18 and Glu 174 of the hormone. In PLR the orientation of this loop is such that the Asp and His residues, in combination with His and Glu from the hormone, form a strong binding site for a zinc atom that links the hormone and the receptor (Figure 13.23b). The presence of zinc increases the affinity of the hormone for the receptor in vitro by a factor of 10,000. As shown by mutagenesis studies His 18 and Glu 174 of the hormone are important for the tight binding to PRL but not to GHR. 

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