Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic E. coli BL-21 and auxotrophic E. coli B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b5 (b5M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic E. coli, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b5M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70-80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.

• Keywords
• E. coli, azidohomoalanine, bio-orthogonal amino acid global substitution, homopropargylglycine, non-canonical amino-acid-containing proteins, photo-methionine,
• MeSH
• Alanine MeSH
• Amino Acids metabolism   MeSH
• Escherichia coli * genetics   metabolism   MeSH
• Methionine * metabolism   MeSH
• Proteins chemistry   MeSH
• Racemethionine metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Alanine MeSH
• Amino Acids MeSH
• azidohomoalanine MeSH Browser
• homopropargylglycine MeSH Browser
• Methionine * MeSH
• Proteins MeSH
• Racemethionine MeSH

Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b₅, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.

• Keywords
• contribution of cytochromes P450 in detoxification of aristolochic acid I in human and rat livers, cytochrome P450-mediated detoxification of aristolochic acid I, molecular modeling, plant nephrotoxin and carcinogen aristolochic acid I,
• MeSH
• Cytochrome P-450 Enzyme Inhibitors pharmacology   MeSH
• Microsomes, Liver drug effects   metabolism   MeSH
• Liver drug effects   metabolism   MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Rats MeSH
• Aristolochic Acids adverse effects   chemistry   metabolism   MeSH
• Humans MeSH
• Activation, Metabolic MeSH
• Inactivation, Metabolic MeSH
• Methylation drug effects   MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Kidney Diseases etiology   metabolism   MeSH
• Oxidation-Reduction drug effects   MeSH
• Cytochrome P-450 Enzyme System chemistry   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• aristolochic acid I MeSH Browser
• Cytochrome P-450 Enzyme Inhibitors MeSH
• Aristolochic Acids MeSH
• Cytochrome P-450 Enzyme System MeSH

Protein-protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8-Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.

• MeSH
• Photochemical Processes MeSH
• Humans MeSH
• Protein Interaction Mapping methods   MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• 14-3-3 Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 14-3-3 Proteins MeSH