ABSTRACT
Diverse proteome reactivity profiles were obtained using small-molecule electrophiles. Based on the cross-reactivity profile, each protein generated a unique reactivity fingerprint. Here, we report the first proteome reactivity signature-based discrimination of 11 bacteria. Perfect differentiation of 11 bacteria can be achieved using 2 benzyl-halide probes.
Subject(s)
Bacteria/isolation & purification , Bacteria/metabolism , Electrophoresis, Polyacrylamide Gel , Proteome/analysis , Fluorescent Dyes/chemistry , Molecular Probes/chemistry , Principal Component Analysis , Proteome/metabolism , Spectrometry, FluorescenceABSTRACT
A mitochondria-trackable fluorophore, CDy2, selectively labels Cys302 in the active site of mitochondrial aldehyde dehydrogenase (ALDH2).
Subject(s)
Aldehyde Dehydrogenase/chemistry , Cysteine/metabolism , Fluorescent Dyes/chemistry , Heterocyclic Compounds, 3-Ring/chemistry , Mitochondria/metabolism , Piperidines/chemistry , Affinity Labels/chemistry , Affinity Labels/metabolism , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Binding Sites , Cysteine/chemistry , Fluorescent Dyes/metabolism , HEK293 Cells , Heterocyclic Compounds, 3-Ring/metabolism , Humans , Mutagenesis, Site-Directed , Piperidines/metabolismABSTRACT
The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce beta-lactamase activity into the alphabeta/betaalpha metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo beta-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat/Km)app of 1.8 x 10(2) (mole/liter)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.
Subject(s)
Directed Molecular Evolution , Protein Engineering , Thiolester Hydrolases/chemistry , Thiolester Hydrolases/metabolism , beta-Lactamases/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Catalytic Domain , Cefotaxime/metabolism , Cefotaxime/pharmacology , Drug Resistance, Bacterial , Escherichia coli/drug effects , Evolution, Molecular , Humans , Hydrophobic and Hydrophilic Interactions , Iron/metabolism , Kinetics , Metals/metabolism , Models, Molecular , Molecular Sequence Data , Point Mutation , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Thiolester Hydrolases/genetics , Zinc/metabolism , beta-Lactamases/chemistryABSTRACT
Quantitative structure-activity relationships (QSAR) analyses were carried out on the SH2-phosphopeptide complexes using multiple linear regressions. The residue-residue interaction energies and cross-correlation coefficients were used as descriptors. Since the number of descriptors was very large (602 for interaction energies and 951 for cross-correlation coefficients), the stepwise addition method was applied for the multiple linear regressions. The residue-residue interaction energies were good descriptors for structure-activity relationships. The high r(2) regression models were achieved by using interaction energy. In addition, the concerted atomic motions, which show the dynamic properties during the SH2-phosphopeptide interaction, were used as descriptors. They were identified by the cross-correlation coefficients for atomic displacement. The best regression model, derived by using four cross-correlation coefficients, gave a high r(2) value of 0.925. This suggests that the dynamic properties showing concerted atomic motions can be used as good descriptors in QSAR study.
Subject(s)
Phosphopeptides/metabolism , src Homology Domains , Amino Acid Sequence , Binding Sites , Models, Molecular , Phosphopeptides/chemistry , Protein Binding , Quantitative Structure-Activity Relationship , Regression Analysis , ThermodynamicsABSTRACT
A comparative molecular field analysis (CoMFA) was carried out to investigate quantitative structure-activity relationships for SH2-binding phosphopeptides. Two alignment rules were applied in our CoMFA model. The phosphopeptide backbone atoms were used for superposition in alignment I and the backbone atoms of peptide-binding residues of SH2-phosphopeptide complexes were used in alignment II to consider the position of phosphopeptides in SH2-binding sites. The higher correlation and predictivity in alignment II (r(2) value of 0.961 and cross-validated r(2) value of 0.682) suggest that the consideration of peptide-binding position at the binding sites gives rise to better results when the ligand-receptor complex structure is considered. In addition, CoMFA contour and electrostatic maps were well accorded with the experimental results in which the replacement of N-terminal residues with an acetyl group reduced the binding affinity. Therefore, the modification of molecular size and charge of phosphopeptides can be carried out based on these contour maps in order to increase binding affinities.
