Pei Jing Pai

Posttranslational modification of CENP-A influences the conformation of centromeric chromatin

Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.

A genetically encoded photocaged Nε-methyl-L-lysine

A photocaged N(epsilon)-methyl-L-lysine has been genetically incorporated into proteins at amber codon positions in Escherichia coli using an evolved pyrrolysyl-tRNA synthetase-pylT pair. Its genetic incorporation and following photolysis to recover N(epsilon)-methyl-L-lysine at physiological pH provide a convenient method for the biosynthesis of proteins with monomethylated lysines at specific sites.

A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli

By overexpressing the C-terminal domain of the ribosomal protein L11 to decrease release factor 1-mediated termination of protein translation, enhanced amber suppression is achieved in E. coli. This enhanced amber suppression efficiency allows the genetic incorporation of three N(epsilon)-acetyl-l-lysines into one GFP(UV) protein in E. coli.

Hepatic phenotype of liver fatty acid binding protein gene-ablated mice

Although the function of liver fatty acid binding protein in hepatic fatty acid metabolism has been extensively studied, its potential role in hepatic cholesterol homeostasis is less clear. Although hepatic cholesterol accumulation was initially reported in L-FABP-null female mice, that study was performed with early N2 backcross generation mice. To resolve whether the hepatic cholesterol phenotype in these L-FABP(-/-) mice was attributable to genetic inhomogeneity, these L-FABP(-/-) mice were further backcrossed to C57Bl/6 mice up to the N10 (99.9% homogeneity) generation. Hepatic total cholesterol accumulation was observed in female, but not male, L-FABP(-/-) mice at all (N2, N4, N6, N10) backcross generations examined. The greater total cholesterol was due to increased hepatic levels of both unesterified (free) cholesterol and esterified cholesterol. Altered hepatic cholesterol accumulation correlated directly with L-FABPs ability to bind cholesterol with high affinity as shown by direct L-FABP binding of fluorescent cholesterol analogs (NBD-cholesterol, dansyl-cholesterol), a photoactivatable cholesterol analog [free cholesterol benzophenone (FCBP)], and free cholesterol (circular dichroism, isothermal titration microcalorimetry). One mole of fluorescent sterol was bound per mole of L-FABP. This was confirmed by photo-cross-linking studies with the photoactivatable cholesterol analog FCBP and by isothermal titration calorimetry with free cholesterol, which showed that L-FABP bound only one sterol molecule per L-FABP molecule. In contrast, the hepatic phenotype of male, but not female, L-FABP(-/-) mice was characterized by decreased hepatic triacylglycerol levels at all backcross generations examined. Taken together, these data support the hypothesis that L-FABP plays a role in physiological regulation of not only hepatic fatty acid metabolism, but also that of hepatic cholesterol.

The de novo engineering of pyrrolysyl-tRNA synthetase for genetic incorporation of L-phenylalanine and its derivatives

Using evolved pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pairs, L-phenylalanine, p-iodo-L-phenylalanine and p-bromo-L-phenylalanine have been genetically incorporated into proteins at amber mutation sites in E. coli.

Genetic incorporation of an aliphatic keto-containing amino acid into proteins for their site-specific modifications.

2-Amino-8-oxononanoic acid has been genetically incorporated into proteins in Escherichia coli by the use of an evolved pyrrolysyl-tRNA synthetase/pylT pair. The direct usage of the exclusive reactivity of the keto group in this amino acid with hydrazide- and alkoxyamine-bearing compounds to site-specifically label proteins under a mild condition close to physiological pH exhibited a very high efficiency.

Evidence for Radical-Mediated Catalysis by HppE: A Study Using Cyclopropyl and Methylenecyclopropyl Substrate Analogues

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that catalyzes the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid ((S)-HPP) in the biosynthesis of the antibiotic fosfomycin. HppE also recognizes (R)-2-hydroxypropylphosphonic acid ((R)-HPP) as a substrate and converts it to 2-oxo-propylphosphonic acid. To probe the mechanisms of these HppE-catalyzed oxidations, cyclopropyl- and methylenecyclopropyl-containing compounds were synthesized and studied as radical clock substrate analogues. Enzymatic assays indicated that the (S)- and (R)-isomers of the cyclopropyl-containing analogues were efficiently converted to epoxide and ketone products by HppE, respectively. In contrast, the ultrafast methylenecyclopropyl-containing probe inactivated HppE, consistent with a rapid radical-triggered ring-opening process that leads to enzyme inactivation. Taken together, these findings provide, for the first time, experimental evidence for the involvement of a C2-centered radical intermediate with a lifetime on the order of nanoseconds in the HppE-catalyzed oxidation of (R)-HPP.

Mechanistic Consequences of Chiral Radical Clock Probes: Analysis of the Mononuclear Non-Heme Iron Enzyme HppE with 2-Hydroxy-3-methylenecyclopropyl Radical Clock Substrates

(S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C–H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.

Efficient Electrophoretic Method to Remove Neutral Additives from Protein Solutions Followed by Mass Spectrometry Analysis

A mass spectrometry (MS)-compatible, isoelectric point-based separation method for removal of neutral additives from protein solutions is described. The separation is performed by electrophoretic migration and trapping using a device referred to as membrane separated wells for isoelectric focusing and trapping (MSWIFT). Electrophoretic separation in the MSWIFT device is fast; the entire process can be carried out in a matter of minutes, and it does not require further sample cleanup prior to MS analysis. Proof-of-concept experiments in which neutral additives (e.g., Triton X-100, Tween 20, poly(ethylene glycol)) are removed from protein solutions using the MSWIFT device followed by MS analysis are described. Coupling the MSWIFT separation with ion mobility MS provides additional separation via the gas phase and assists in achieving higher quality ESI mass spectra when small amounts of additives remain in solution.