A spectroelectrochemical method has been used to determine the reduction potential of the copper site in wild-type and 22 mutant forms of azurin from Pseudomonas aeruginosa at 25 "C and in the range pH 4-8; the effect of buffers and ionic strength on the potentials has also been studied. Amino-acid residues changed include Metl21, which provides an S atom at a distance of about 0.3 nm from the metal, some amino acids in the hydrophobic patch, other residues believed to be important in electron transfer with physiological partners and some internal amino acids. The observed potentials span a range of about 300 mV. In all cases the potentials increase with decreasing pH, but the pK, values describing the pH dependence are essentially unchanged except in three mutants, where they change by pH 0.6-1.1 (up in one and down in two). The largest potential changes were found in some Met121 mutants, at which position large hydrophobic residues raise the potential, whereas negatively charged residues lower it; a decreased potential is also found in the Met121 + End mutant, which probably has H,O coordinated to the metal. Gly45 has its carbonyl group coordinated to copper, but the potential of Gly45 3 Ala is close to that of the wild type. Some substitutions in the hydrophobic patch cause an increase in the potential, whereas substitutions involving His35 and Glu91 do not result in significant changes. No single mechanism for tuning the potential of the copper site can be discerned, but in many cases there are probably indirect effects of the protein conformation causing changes in metal-ligand interactions.Azurin belongs to a family of small blue-copper proteins which function in electron-transfer chains in plants and bacteria (Adman, 1985). These proteins have unique spectral properties, such as intense absorbance bands around 600 nm and a narrow hypefine splitting in the CuZ+ EPR signal, and they also have unusually high reduction potentials among Cu2+ complexes. A perplexing fact is, however, that these potentials can vary by more than 0.5 V between members of the family despite the fact that the spectroscopic properties are relatively constant. Since the intense blue color and specific EPR signal are properties of the oxidized proteins, it has been suggested that proteins with a high reduction potential have a preferential stabilization of the Cu+ form (Gray and Malmstrom, 1983). To test this hypothesis, or other possible mechanisms for tuning the potentials, is one of the purposes of the program in site-directed mutagenesis of which the study described here is part.We have prepared mutant forms of azurin with substitutions of a metal ligand (Metl21) (Karlsson et al., 1991), of surface residues, some believed important in the interactions with electron-transfer partners , and also of some residues localized more internally. In this study we report the determination by thin-layer spectroelectrochemisCorrespondence to T. Vanngird,

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We have made an evaluation of mutation detection techniques for their abilities to detect mosaic mutations. In this study, Sanger sequencing, single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HD), protein truncation test (PTT), and denaturating high-performance liquid chromatography (DHPLC) were compared with parallel sequencing. In total DNA samples from nine patients were included in this study. Mosaic mutations were artificially constructed from seven of these samples, which were from heterozygote mutation carriers with the mutant allele present at 50%. The mutations analyzed were as follows: c.646C>T, c.2626C>T, c.2828C>A, c.1817_1818insA, c.2788dupA, c.416_419delAAGA, and c.607delC in the APC gene. The lowest degree of mutant alleles detected with SSCP/HD and DHPLC varied between 5% and 25%, and between 15% and 50% for Sanger sequencing. Three of the mutations were analyzed with PTT with considerable variations in detection levels (from 10 to 100%). Using parallel sequencing a detection frequency down to 1% was reached, but to achieve this high sensitivity sufficient coverage was required. Two patients with natural mosaic mutations were also included in this study. These two mutations had previously been identified with Sanger sequencing (NF2 c.1026_1027delGA) and SSCP/HD (APC c.2700_2701delTC). In conclusion, all the evaluated methods are applicable for mosaic mutation screening even though combinations of the conventional methods should be used to reach an adequate sensitivity. Sanger sequencing alone is not sensitive enough to detect low mosaic levels. Parallel sequencing seems to be the ultimate choice but the possibilities to use this technique is today limited by its complexity, economics, and availability of instruments.

Cassette mutagenesis was used to exchange the suggested copper ligand Met121 in azurin to all other amino acids, and a stop codon. The mutant proteins were characterized by optical absorption spectroscopy and EPR. At low pH, all mutants exhibit the characteristics of a blue type 1 copper protein, indicating that methionine is not needed to create a blue copper site. At high pH, the Glu121 and the Lys121 mutants constitute a new form of protein-bound copper that is outside the range of type 1 copper.

The structural gene for the blue copper protein azurin from Pseudomonas aeruginosa has been subcloned in different expression plasmid vectors. The highest yield of expression was obtained when the gene with its native ribosome-binding site was placed downstream of the lac promoter in plasmid pUCI8. The protein is exported to the periplasmic space in Escherichia coli and the amount corresponds to 27% of the total protein content in the periplasmic space. The preprotein is cleaved correctly according to N-terminal sequencing of the purified protein. Azurin has been purified in large amounts and is spectroscopically indistinguishable from the protein purified from P. aeruginosa.

The latter mutant shows a 2-fold slower mtrdcomplcu ET to photosystem I (PSI) as expected from the decreased driving forceThe affinity for PSI IS unaffected for this mutant but IS drabtlcally decreased for Pc It IS concluded that the hydrophobic patch IS more important for the ET to PSI Electron transfer, Flash photolysls, Pldstocyanm, Photosynthesls. Photosystem 1, Sltc-duzctcd rnutdgenesrs

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