Identifying Protein-Drug Interactions in Cell Lysates Using Histidine Hydrogen Deuterium Exchange.

Identifying the targets of a drug is critical to understand the mechanism of action and predicts possible side effects. The conventional approach is capturing interacting proteins by affinity purification. However, it requires drugs to be immobilized to a solid support or derivatized with chemical moieties used for pulling down interacting proteins. Such covalent modifications to drugs may mask a critical recognition site for or alter the binding affinity to their targets. To overcome the drawback, several methods that do not require covalent modifications to drugs have been developed. These methods identify targets by detecting proteins whose thermodynamic stability is enhanced in the presence of drugs. Although the utility of these methods has been demonstrated, the difficulty in identifying low abundant targets is the common problem of these methods. We have developed a new target identification method that increases the likelihood of identifying low abundant targets. The method uses histidine-hydrogen deuterium exchange (His-HDX) as a readout technique to probe the changes in protein stability induced by drugs. The workflow involves incubating cell lysates in various concentrations of a protein denaturant in the presence and absence of a drug in D2O followed by digestion of the proteins, enrichment of His-containing peptides, and analysis of the enriched His-peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The developed method was successfully applied to identify the interaction between endogenously expressed MAPK14 and its inhibitor in HEK293 cell lysates. The implementation of selective enrichment of histidine-containing peptides in the workflow was a key that enabled identifying the MAPK14-inhibitor interaction.

Combination of N149S and D171G mutations in Aeromonas caviae polyhydroxyalkanoate synthase and impact on polyhydroxyalkanoate biosynthesis.

Aeromonas caviae polyhydroxyalkanoate synthase (PhaC(Ac)) is an important biocatalyst for the synthesis of practically useful two-component polyhydroxyalkanoate copolymer, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. In a previous study, two PhaC(Ac) mutants that have a single amino acid substitution of either asparagine 149 by serine (N149S) or aspartate 171 by glycine (D171G) were isolated as higher active enzymes by means of evolutionary engineering. In this study, the synergistic effects of N149S and D171G double mutation (NSDG) in PhaC(Ac) on polyhydroxyalkanoate biosynthesis were investigated in recombinant Ralstonia eutropha. The PhaC(Ac) NSDG mutant showed enhanced incorporation of longer 3-hydroxyalkanoate (3HA) units into the polyhydroxyalkanoate copolymer from octanoate (3HA fraction: 18.5 mol%) and soybean oil (5.4 mol%) as a carbon source. Besides, the NSDG mutant synthesized P(3HB) homopolymer with a very high molecular weight (M(w)=368 x 10(4)) when fructose was used as a carbon source. Thus, a combination of the beneficial mutations synergistically altered enzymatic properties, leading to synthesis of a polyhydroxyalkanoate copolymer with enhanced 3HA fraction and increased molecular weight.

Variation in copolymer composition and molecular weight of polyhydroxyalkanoate generated by saturation mutagenesis of Aeromonas caviae PHA synthase.

Amino acid substitutions at two residues downstream from the active-site histidine of polyhydroxyalkanoate (PHA) synthases are effective for changing the composition and the molecular weight of PHA. In this study, saturation mutagenesis at the position Ala505 was applied to PHA synthase (PhaCAc) from Aeromonas caviae to investigate the effects on the composition and the molecular weight of PHA synthesized in Ralstonia eutropha. The copolymer composition and molecular weight of PHA were varied by association with amino acid substitutions. There was a strong relationship between copolymer composition and PHA synthase activity of the cells. This finding will serve as a rationale for producing tailor-made PHAs.