ABSTRACT
Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of δ-thiol-L-lysine (7) and δ-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.
Subject(s)
Protein Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ubiquitination , Amino Acyl-tRNA Synthetases/metabolism , Binding Sites , Lysine/analogs & derivatives , Lysine/metabolism , Methanosarcina barkeri/enzymology , Recombinant Proteins/genetics , Substrate SpecificityABSTRACT
Lysine methylation is an important post-translational modification of histone proteins that defines epigenetic status and controls heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair, and transcriptional regulation. Despite considerable efforts by chemical biologists to synthesize modified histones for use in deciphering the molecular role of methylation in these phenomena, no general method exists to synthesize proteins bearing quantitative site-specific methylation. Here we demonstrate a general method for the quantitative installation of N(epsilon)-methyl-L-lysine at defined positions in recombinant histones and demonstrate the use of this method for investigating the methylation dependent binding of HP1 to full length histone H3 monomethylated on K9 (H3K9me1). This strategy will find wide application in defining the molecular mechanisms by which histone methylation orchestrates cellular phenomena.
Subject(s)
Histones/genetics , Lysine/analogs & derivatives , Recombinant Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Histones/biosynthesis , Histones/metabolism , Lysine/genetics , Lysine/metabolism , Methylation , Mutagenesis, Site-Directed/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolismABSTRACT
We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNA(CUA) pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-L-lysine, containing a carbon-carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-L-lysine, containing an azido group, into recombinant proteins in Escherichia coli. Proteins containing the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddition reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chemically orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topology and structure by intramolecular orthogonal conjugation reactions.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Archaeal Proteins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli/genetics , Methanosarcina barkeri/enzymology , Protein Engineering/methods , Recombinant Proteins/genetics , Alkynes/chemistry , Alkynes/metabolism , Amino Acyl-tRNA Synthetases/genetics , Archaeal Proteins/metabolism , Azides/chemistry , Azides/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Myoglobin/chemistry , Myoglobin/genetics , Myoglobin/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
The development of a novel method to attenuate bacterial virulence is reported, which is based upon the use of designed transition-state analogues to select human catalytic antibodies capable of degrading bacterial quorum-sensing molecules.
Subject(s)
Acyl-Butyrolactones/chemistry , Anti-Bacterial Agents/metabolism , Antibodies, Catalytic/metabolism , Quorum Sensing , Sulfones/chemistry , Bacterial Proteins/antagonists & inhibitors , HydrolysisABSTRACT
A few Mannich ketones of piperazinyl oxazolidinone derivatives have been synthesized and their antibacterial activity in various Gram-positive organisms such as Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis were evaluated by MIC determination. Compound 12 showed comparable activity (MIC) to linezolid and superior to eperezolid.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Ketones/chemical synthesis , Oxazolidinones/chemical synthesis , Piperazines/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/drug effects , Enterococcus faecalis/drug effects , Ketones/chemistry , Ketones/pharmacology , Microbial Sensitivity Tests , Oxazolidinones/chemistry , Oxazolidinones/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus epidermidis/drug effectsABSTRACT
Synthesis of a number of 4,5,6,7-tetrahydro-thieno[3,2-c]pyridine substituted oxazolidinones have been reported. They have been screened against a panel of Gram-positive pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. A SAR has been developed. Compound 15 showed comparable activity (MIC) to linezolid and superior to eperezolid.
