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Abstract
A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.
Highlights
Introduction of Mutations intoPseudomonas aeruginosa holocytochrome c551 (PA c551) Gene—Two methods for sitedirected mutagenesis were used to introduce a series of mutations in PA c551
Expression of PA c551 in the Periplasm of E. coli—The wildtype PA c551 protein expressed in E. coli JCB7120 strain was fully recovered in the cold osmotic shock fluid containing the periplasmic protein fraction, but not in the membrane and cytoplasmic fractions
The N-terminal amino acid sequence of the wild-type PA c551 protein expressed in the E. coli periplasm was determined as Glu-Asp-Pro-Glu-Val-Leu-Phe-Lys-Asn-Lys-Gly, which was identical to that of the authentic protein purified from the native organism
Summary
Bacterial Strain, Plasmids, and Growth Condition—The EcoRI-PstI gene fragment CP1, encoding the 22-amino acid signal sequence and. The pKPA1-based plasmids carrying mutated PA c551 gene fragments (see below) were transformed by standard methods into E. coli JCB7120 strain in which the expression of c-type cytochromes is unusually high.. The transformed E. coli cells were grown anaerobically in minimal media in the presence of glycerol, nitrite, and fumarate [19] supplemented with casamino acid (2 mg/ml), tryptophan (20 g/ml), and ampicillin (50 g/ml) at 37 °C for the production of wild-type and mutant PA c551 proteins. The N-terminal amino acid sequence of the purified wild-type PA c551 expressed in the E. coli periplasm was determined by automatic sequencer (Applied Biosystems model 470A). Protein Thermostability—The wild-type and mutant PA c551 proteins (10 g/ml protein concentration in water, pH 5.0 adjusted with HCl) were subjected to the thermal melting profile analysis by monitoring the changes of CD spectra at 222 nm as described previously [15] with slight modifications. All other chemicals used were of the highest grade commercially available
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