Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
- MeSH
- Affinity Labels chemical synthesis chemistry radiation effects MeSH
- Aspartic Acid Endopeptidases antagonists & inhibitors chemistry MeSH
- Biotin chemistry MeSH
- Diazomethane analogs & derivatives chemical synthesis radiation effects MeSH
- Fluoresceins chemistry MeSH
- Fluorescent Dyes chemistry MeSH
- Glutamate Carboxypeptidase II antagonists & inhibitors chemistry MeSH
- Mass Spectrometry methods MeSH
- Enzyme Inhibitors chemical synthesis chemistry radiation effects MeSH
- Microscopy, Confocal methods MeSH
- Polymethacrylic Acids chemistry MeSH
- Humans MeSH
- Membrane Proteins antagonists & inhibitors chemistry MeSH
- Cell Line, Tumor MeSH
- Proteomics methods MeSH
- Serine Endopeptidases chemistry MeSH
- Ultraviolet Rays MeSH
- Gelatinases antagonists & inhibitors chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Studium proteinů a zvláště jejich 3D struktury či protein -proteinových interakcí hraje v dnešním biochemickém výzkumu nezanedbatelnou roli. Řada alternativních metod tohoto výzkumu (např. PIXL, FRET) využívá biotechnologické postupy pro zavedení nepřirozených aminokyselin či jejich strukturních analogů do proteinové sekvence během jejich rekombinantní přípravy. Předkládaná práce uvádí několik biotechnologických přístupů inkorporace foto -methioninu (pMet, L-2-amino-5,5-azi -hexanová kyselina) do sekvence dvou modelových savčích proteinů.
The study of proteins and especially their 3D structure or protein -protein interactions plays significant role in contemporary biochemical research. Many alternative methods of the research (e.g. PIXL, FRET) employing different biotechnology techniques to introduce the non -natural amino acids or their structural analogues within protein sequence during its recombinant expression. This study presents several biotechnology approaches to introduce photo -methionine (pMet, L-2-amino-5,5-azi -hexanoic acid) into the sequence of two model mammalian proteins.
- Keywords
- světlem iniciované síťování, foto-methionin,
- MeSH
- Amino Acyl-tRNA Synthetases metabolism MeSH
- Amino Acids chemistry metabolism MeSH
- Biotechnology MeSH
- Diazomethane MeSH
- Protein Conformation * MeSH
- Protein Interaction Mapping * MeSH
- Methionine MeSH
- Protein Processing, Post-Translational * MeSH
- Proteins chemistry MeSH
- Protein Biosynthesis MeSH
- In Vitro Techniques MeSH
- Imaging, Three-Dimensional MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin.
- MeSH
- Cysteine Endopeptidases metabolism drug effects MeSH
- Diazomethane analogs & derivatives pharmacology MeSH
- Financing, Organized MeSH
- Chromatography, Gel MeSH
- Protease Inhibitors pharmacology MeSH
- Keratins metabolism MeSH
- Collagen metabolism MeSH
- Hydrogen-Ion Concentration MeSH
- Leucine analogs & derivatives metabolism MeSH
- Schistosoma mansoni enzymology MeSH
- Schistosomatidae enzymology MeSH
- Binding Sites MeSH
- Gelatin metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Comparative Study MeSH
- MeSH
- Biochemical Phenomena MeSH
- Biochemistry MeSH
- Diazomethane MeSH
- Cathepsins metabolism MeSH
- Rabbits MeSH
- Culture Techniques MeSH
- Methods MeSH
- Nucleic Acids metabolism MeSH
- Pneumonia chemically induced metabolism MeSH
- Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Female MeSH
- Animals MeSH
