Protein Engineering on Azurin. Expression Mutagenesis and Characterisation of Copper Site Mutants
Doktorsavhandling, 1992

Azurin belongs to a family of small blue copper proteins or cupredoxins which participate in electron transfer processes in plants and bacteria. The type 1 copper site in these proteins is characterised by an intense blue colour, a narrow hyperfine coupling in the EPR signal and, generally, a high reduction potential. A common feature in the cupredoxin structure is the eight stranded b-barrel and a copper binding site situated in a loop between two b-strands. Three of the four copper ligands, two histidines and one cysteine, are conserved in all cupredoxins, whereas the fourth ligand, a methionine, is exchanged in stellacyanin. The gene encoding azurin from Pseudomonas aeruginosa has been expressed in large amounts in Escherichia coli. Cassette mutagenesis was used to exchange the methionine ligand for all other amino acids, including a stop codon. The properties of the mutant proteins were investigated by optical and EPR spectroscopy. The effect of the amino acid exchange on the reduction potential was studied, XAFS was used to analyse the structure of the copper site and the X-ray structure was determined for the Met121Glu mutant protein. It was concluded that the ligand methionine is not needed to create a type 1 copper site. The exchange of the methionine results in mutant proteins with large changes in spectroscopic and redox properties. The introduction of polar residues gives rise to spectroscopic properties similar to properties found in stellacyanin or pseudoazurin. A large increase in reduction potential is observed when hydrophobic residues are introduced, whereas a decrease is observed when the copper ion is solvent exposed. The introduction of a negative group directly coordinating the copper is accompanied by a change in the copper site geometry, a decrease in reduction potential and a drastic change in the spectroscopic properties.

EPR spectroscopy

cassette mutagenesis

optical spectroscopy

amino acid exchange

Escherichia coli

electron transfer

reduction

azurin

Författare

B Göran Karlsson

Institutionen för biokemi och biofysik

Ämneskategorier

Industriell bioteknik

ISBN

91-7032-790-4

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 917