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Domain-swapped dimer of Pseudomonas aeruginosa cytochrome c551: structural insights into domain swapping of cytochrome c family proteins.

Nagao S, Ueda M, Osuka H, Komori H, Kamikubo H, Kataoka M, Higuchi Y, Hirota S - PLoS ONE (2015)

Bottom Line: The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping.The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins.These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.

ABSTRACT
Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c551 (PA cyt c551), and Hydrogenobacter thermophilus cytochrome c552 (HT cyt c552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met-heme coordination significantly compared to the monomer. HT cyt c552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

No MeSH data available.


CD spectra and small angle X-ray scattering curves of WT and M61A PA cyt c551.(A) CD spectra of oxidized monomeric WT (red) and M61A (green) PA cyt c551. Measurement conditions: Sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature. (B) Small angle X-ray scattering curves of oxidized monomeric WT (red) and M61A (green) PA cyt c551 shown by Kratky plots. The intensities are normalized at their maximum intensities. Measurement conditions: sample concentration, 500 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 20°C.
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pone.0123653.g004: CD spectra and small angle X-ray scattering curves of WT and M61A PA cyt c551.(A) CD spectra of oxidized monomeric WT (red) and M61A (green) PA cyt c551. Measurement conditions: Sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature. (B) Small angle X-ray scattering curves of oxidized monomeric WT (red) and M61A (green) PA cyt c551 shown by Kratky plots. The intensities are normalized at their maximum intensities. Measurement conditions: sample concentration, 500 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 20°C.

Mentions: We replaced the heme-ligating Met61 of PA cyt c551 with Ala (M61A PA cyt c551) to investigate the effect of Met61 on oligomerization. The Soret band of oxidized monomeric WT PA cyt c551 at 409 nm blue shifted to 401 nm in the oxidized monomeric M61A PA cyt c551 spectrum (S7 Fig). The intensities of the negative 208-nm and 222-nm CD bands of oxidized M61A PA cyt c551 decreased by about 10% from those of the corresponding bands of oxidized WT PA cyt c551 (Fig 4A), indicating that the α-helical content of M61A PA cyt c551 decreased slightly compared to that of the WT protein. The radii of gyration were obtained as 13.7 and 13.9 Å for WT and M61A PA cyt c551, respectively, by SAXS measurements (Fig 4B). Although the size of the global structure of PA cyt c551 did not change significantly by the removal of Met61, the secondary structures were slightly perturbed (Fig 4). It has been reported that carboxylmethylation of Met61 of PA cyt c551 destabilizes its folded state [48], indicating that the α-helical structure of PA cyt c551 is stabilized by the Met–heme coordination. The amount of dimer produced by the treatment with ethanol decreased to less than 5% and no trimer or tetramer was detected for M61A PA cyt c551 (S8 Fig). These results indicate that the removal of heme-ligating Met in PA cyt c551 suppressed formation of oligomers by domain swapping.


Domain-swapped dimer of Pseudomonas aeruginosa cytochrome c551: structural insights into domain swapping of cytochrome c family proteins.

Nagao S, Ueda M, Osuka H, Komori H, Kamikubo H, Kataoka M, Higuchi Y, Hirota S - PLoS ONE (2015)

CD spectra and small angle X-ray scattering curves of WT and M61A PA cyt c551.(A) CD spectra of oxidized monomeric WT (red) and M61A (green) PA cyt c551. Measurement conditions: Sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature. (B) Small angle X-ray scattering curves of oxidized monomeric WT (red) and M61A (green) PA cyt c551 shown by Kratky plots. The intensities are normalized at their maximum intensities. Measurement conditions: sample concentration, 500 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 20°C.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390240&req=5

pone.0123653.g004: CD spectra and small angle X-ray scattering curves of WT and M61A PA cyt c551.(A) CD spectra of oxidized monomeric WT (red) and M61A (green) PA cyt c551. Measurement conditions: Sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature. (B) Small angle X-ray scattering curves of oxidized monomeric WT (red) and M61A (green) PA cyt c551 shown by Kratky plots. The intensities are normalized at their maximum intensities. Measurement conditions: sample concentration, 500 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 20°C.
Mentions: We replaced the heme-ligating Met61 of PA cyt c551 with Ala (M61A PA cyt c551) to investigate the effect of Met61 on oligomerization. The Soret band of oxidized monomeric WT PA cyt c551 at 409 nm blue shifted to 401 nm in the oxidized monomeric M61A PA cyt c551 spectrum (S7 Fig). The intensities of the negative 208-nm and 222-nm CD bands of oxidized M61A PA cyt c551 decreased by about 10% from those of the corresponding bands of oxidized WT PA cyt c551 (Fig 4A), indicating that the α-helical content of M61A PA cyt c551 decreased slightly compared to that of the WT protein. The radii of gyration were obtained as 13.7 and 13.9 Å for WT and M61A PA cyt c551, respectively, by SAXS measurements (Fig 4B). Although the size of the global structure of PA cyt c551 did not change significantly by the removal of Met61, the secondary structures were slightly perturbed (Fig 4). It has been reported that carboxylmethylation of Met61 of PA cyt c551 destabilizes its folded state [48], indicating that the α-helical structure of PA cyt c551 is stabilized by the Met–heme coordination. The amount of dimer produced by the treatment with ethanol decreased to less than 5% and no trimer or tetramer was detected for M61A PA cyt c551 (S8 Fig). These results indicate that the removal of heme-ligating Met in PA cyt c551 suppressed formation of oligomers by domain swapping.

Bottom Line: The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping.The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins.These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.

ABSTRACT
Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c551 (PA cyt c551), and Hydrogenobacter thermophilus cytochrome c552 (HT cyt c552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met-heme coordination significantly compared to the monomer. HT cyt c552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

No MeSH data available.