Structural characteristics of the hydrophobic patch of azurin and its interaction with p53: a site-directed spin labeling study.
Site-directed spin labeling (SDSL) is a powerful tool for monitoring protein structure, dynamics and conformational changes. In this study, the domain-specific properties of azurin and its interaction with p53 were studied using this technique. Mutations of six residues, that are located in the hydrophobic patch of azurin, were prepared and spin labeled. Spectra of the six azurin mutants in solution showed that spin labeled residues 45 and 63 are in a very restricted environment, residues 59 and 65 are in a spacious environment and have free movement, and residues 49 and 51 are located in a relatively closed pocket. Polarity experiments confirmed these results. The changes observed in the spectra of spin labeled azurin upon interaction with p53 indicate that the hydrophobic patch is involved in this interaction. Our results provide valuable insight into the topographic structure of the hydrophobic domain of azurin, as well as direct evidence of its interaction with p53 in solution via the hydrophobic patch. Cytotoxicity studies of azurin mutants showed that residues along the hydrophobic patch are important for its cytotoxicity.
Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
Chao
C
Xu
Junjie
J
Yin
Baolu
B
Zhao
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004578:Electron Spin Resonance Spectroscopy; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
D000970:Antineoplastic Agents; D050505:Mutant Proteins; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D016159:Tumor Suppressor Protein p53; D001400:Azurin