Combining free energy calculations with tailored enzyme activity assays to elucidate substrate binding of a phospho-lysine phosphatase.

Studying enzymes that are involved in the regulation of dynamic post-translational modifications (PTMs) is of key importance in proteomics research. Such investigations can be particularly challenging when the modification itself is intrinsically labile. In this article, we elucidate the enzymatic activity of (LHPP) towards different - and -phosphorylated peptides by a combined experimental and computational approach.

Synthesis and Evaluation of Non-Hydrolyzable Phospho-Lysine Peptide Mimics.

Invited for the cover of this issue is the group of Christian P. R. Hackenberger at the Leibniz-Forschungsinstitut für Molekulare Pharmakologie and the Humboldt-Universität zu Berlin.

Synthesis and Evaluation of Non-Hydrolyzable Phospho-Lysine Peptide Mimics.

The intrinsic lability of the phosphoramidate P-N bond in phosphorylated histidine (pHis), arginine (pHis) and lysine (pLys) residues is a significant challenge for the investigation of these post-translational modifications (PTMs), which gained attention rather recently. While stable mimics of pHis and pArg have contributed to study protein substrate interactions or to generate antibodies for enrichment as well as detection, no such analogue has been reported yet for pLys. This work reports the synthesis and evaluation of two pLys mimics, a phosphonate and a phosphate derivative, which can easily be incorporated into peptides using standard fluorenyl-methyloxycarbonyl- (Fmoc-)based solid-phase peptide synthesis (SPPS).

Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations.

In recent years, labile phosphorylation sites on arginine, histidine, cysteine, and lysine as well as pyrophosphorylation of serine and threonine have gained more attention in phosphoproteomic studies. However, the analysis of these delicate posttranslational modifications via tandem mass spectrometry remains a challenge. Common fragmentation techniques such as collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) are limited due to extensive phosphate-related neutral loss.

Chemical Approaches to Investigate Labile Peptide and Protein Phosphorylation.

Protein phosphorylation is by far the most abundant and most studied post-translational modification (PTM). For a long time, phosphate monoesters of serine (pSer), threonine (pThr), and tyrosine (pTyr) have been considered as the only relevant forms of phosphorylation in organisms. Recently, several research groups have dedicated their efforts to the investigation of other, less characterized phosphoamino acids as naturally occurring PTMs.

Maximizing Output in RNA-Programmed Peptidyl-Transfer Reactions.

A chemical reaction that is triggered by a specific RNA molecule might provide opportunities for the design of artificial feedback loops. We envision a peptidyl transfer reaction in which mRNA encoding an antiapoptotic protein would instruct the synthesis of apoptosis-inducing peptides. In this study, we used the RNA-programmed synthesis of a 16-mer peptide derived from the BH3 domain of the protein Bak, which inhibits the antiapoptotic protein Bcl-x .

Development of a novel FRET probe for the real-time determination of ceramidase activity.

Fretful novelty: We developed two novel doubly labelled fluorescent ceramide analogues that exhibit significant FRET and undergo hydrolysis by ceramidases. We present a fluorescent sphingolipid FRET probe that allows homogeneous ratiometric determination of enzyme activity in real-time.