Ulf Reimer

Mapping the stereospecificity of peptidyl prolyl cis/trans isomerases.

The stereospecificity of peptidyl prolyl cis/trans isomerases (PPIases) was studied using tetrapeptide substrate analogs in which one amino acid residue was replaced by the cognate d‐amino acid in various positions of the peptide chain. Reversed stereocenters around proline markedly increased the rate of the spontaneous trans to cis isomerization of the prolyl bond whereas cis to trans isomerizations were less sensitive. PPIases like human cyclophilin18, human FKBP12, Escherichia coli parvulin10 and the PPIase domain of E. coli trigger factor exhibited stereoselectivity demanding at the P1 to P2′ position of the substrate chain. The discriminating factor for stereoselectivity was the lack of formation of the Michaelis complexes of the diastereomeric substrates. However, d‐alanine at the P1 position preserved considerable affinity to the active site, and largely prevented activation of the catalytic machinery for all PPIases investigated.

Peptide Microarrays for Profiling of Epigenetic Targets

Posttranslational modifications (PTMs) are playing a major role in epigenetics, signal transduction, and metabolic processes. We developed a histone peptide microarray presenting more than 3,800 peptides derived from all human histone isoforms. These peptides display most abundant PTMs, such as lysine acylation, lysine/arginine methylation and serine/threonine/tyrosine phosphorylation, and their various combinations. We demonstrate that this histone peptide microarray enables the comprehensive characterization of binding specificities of both antibodies and bromodomains which are claimed to be specific for acylation of lysine side chains. Moreover, we identified histone peptide substrates for protein kinase Wee1 and NAD+-dependent deacetylase sirtuin 3. We were able to show that substrate recognition is influenced by additional side-chain modifications within the substrate sequence. In summary, histone peptide microarrays represent a valuable research tool for the characterization of binding specificities, identification of enzyme substrates, and detection of crosstalk between various PTMs in histones.