Gene synthesis, expression, and mutagenesis of azurin

Citation

Chang, Thomas Kyu-Young (1991) Gene synthesis, expression, and mutagenesis of azurin. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/795e-hp65. https://resolver.caltech.edu/CaltechETD:etd-06142007-132218

Abstract

A synthetic gene for the blue copper protein Pseudomonas aeruginosa azurin has been constructed using a novel, stepwise method. This method is designed to synthesize a gene of any size reliably by building only controlled amounts of the gene in each step. Another advantage of this method is that the intermediate plasmids, which are typically present in picomolar amounts, can be amplified through plasmid preparation for editing. Using this approach, the azurin gene was synthesized in five steps.

Both the synthetic genes for azurin and Populus nigra, var italica plastocyanin have been expressed in E. coli. These expressions have been achieved by using a synthetic Shine-Dalgano sequence and the signal sequence for azurin, which directs the transport of the expressed proteins to the periplasmic space of E. coli. The membrane translocation not only facilitates the purification of azurin and plastocyanin but also seems to be required for the proper folding of these proteins. In contrast to these successful expressions, earlier efforts to express plastocyanin in the cytoplasm of E. coli, either directly or as a fusion protein, have been unsuccessful at yielding folded plastocyanin.

Site-saturation cassette mutagenesis was performed in azurin at Methionine 121, one of the four ligands to the copper. Variants that contain each of the other nineteen amino acids as well as the amber stop codon have been identified. Surprisingly, all the variants are stable, as judged by Western blot. Furthermore, all mutants that have been isolated at this position (Asn, Asp, Gly, His, Ile, Leu, Val) have the characteristic blue absorption near 600 nm. Despite such similarity with the wild-type azurin, these mutants seem to have a more flexible copper center. They can lose or incorporate copper at a faster rate than the wild-type protein.

These results, along with those from EPR experiments, suggest that while Methionine 121 is important in giving stability to the copper center and in tuning the redox potential, its contribution to azurin's spectroscopic properties is small when copper is coordinated to the site. Its spectroscopic contribution apparently becomes more significant when nickel or cobalt resides in the copper site, for the electronic spectra of these derivatives differ markedly from that of azurin with copper. It is likely that the copper centers of the mutants are flexible enough to accommodate the preferred geometries of nickel and cobalt, whereas it is more rigid in the wild-type. Finally, several mutants of azurin are proposed that are designed to probe systematically specific aspects of the biological electron transfer mechanism.

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