ABSTRACT
Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins is valuable for both scientific and biomedical applications. To expand the structural diversity of available ncAAs and to reduce the burden of chemically synthesizing them, we have developed a general and simple biosynthetic method for genetically encoding novel ncAAs into recombinant proteins by feeding cells with economical commercially available or synthetically accessible aromatic thiols. We demonstrate that nearly 50 ncAAs with a diverse array of structures can be biosynthesized from these simple small-molecule precursors by hijacking the cysteine biosynthetic enzymes, and the resulting ncAAs can subsequently be incorporated into proteins via an expanded genetic code. Moreover, we demonstrate that bioorthogonal reactive groups such as aromatic azides and aromatic ketones can be incorporated into green fluorescent protein or a therapeutic antibody with high yields, allowing for subsequent chemical conjugation.
Subject(s)
Amino Acids/biosynthesis , Archaeal Proteins/metabolism , Escherichia coli Proteins/metabolism , Sulfhydryl Compounds/metabolism , Amino Acids/chemistry , Amino Acids/genetics , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Genetic Code , Methanococcus/chemistry , Molecular Structure , Sulfhydryl Compounds/chemistryABSTRACT
In this paper, the interactions of pepsin with CYD (cytidine) or nucleoside analogs, including FNC (2'-deoxy-2'-ß-fluoro-4'-azidocytidine) and CMP (cytidine monophosphate), were investigated by fluorescence, UV-visible absorption and synchronous fluorescence spectroscopy under mimic physiological conditions. The results indicated that FNC (CYD/CMP) caused the fluorescence quenching by the formation of complex. The binding constants and thermo-dynamic parameters at three different temperatures were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize the complex. The F atom in FNC might weaken the binding of nucleoside analog to pepsin. Results showed that CYD was the strongest quencher and bound to pepsin with higher affinity.
Subject(s)
Azides/metabolism , Deoxycytidine/analogs & derivatives , Molecular Docking Simulation , Pepsin A/metabolism , Azides/chemistry , Deoxycytidine/chemistry , Deoxycytidine/metabolism , Energy Transfer , Pepsin A/chemistry , Protein Binding , Protein Conformation , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , TemperatureABSTRACT
The binding of 4'-azido-2'-deoxyfluoroarabinocytidine (FNC) or analogs (cytidine and 5'-cytidylate monophosphate) to bovine serum albumin (BSA) was investigated by fluorescence, UV-vis absorption spectroscopy and molecular modeling. The three compounds quenched the intrinsic fluorescence of BSA and the results revealed the presence of static quenching mechanism. The positive ΔH and positive ΔS for the systems suggested that the hydrophobic forces stabilized the interaction between the compounds and protein. Results also showed that FNC was the weakest quencher.
Subject(s)
Cytidine/analogs & derivatives , Cytidine/metabolism , Deoxycytidine/analogs & derivatives , Models, Molecular , Serum Albumin, Bovine/metabolism , Animals , Cattle , Cytidine/chemistry , Deoxycytidine/chemistry , Deoxycytidine/metabolism , Energy Transfer , Kinetics , Molecular Docking Simulation , Protein Binding , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , TemperatureABSTRACT
The interactions between pepsin and four alkaloids, including caffeine (Caf), aminophylline (Ami), acefylline (Ace), diprophylline (Dip), were investigated by fluorescence, UV-visible absorption, resonance light scattering, synchronous fluorescence spectroscopy and 3D spectroscopy under mimic physiological conditions. The results revealed that Caf (Ami/Ace/Dip) caused the fluorescence quenching of pepsin by the formation of Caf (Ami/Ace/Dip)-pepsin complex. The binding constants and thermodynamic parameters at three different temperatures, the binding locality and the binding power were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize the complex. Results showed that aminophylline was the stronger quencher and bound to pepsin with higher affinity than other three alkaloids.
