Synthetic Receptors for the Recognition and Discrimination of Post‐Translationally Methylated Lysines

Abstract

Post-translational modifications (PTMs) describe the chemical alteration of proteins after their biosynthesis in ribosomes. PTMs play important roles in cell biology, including the regulation of gene expression, cell-cell interactions and the development of different diseases. A prominent class of PTMs is the side-chain methylation of lysine. For the analysis and discrimination of differently methylated lysines, antibodies are widely used, although methylated peptide and protein targets are known to be particularly difficult to differentiate by antibody-based affinity reagents; an additional challenge can be batch-to-batch reproducibility. The application of mass spectrometry techniques for methyllysine discrimination requires a complex sample preparation procedure and is not suited for working in cells. The desire to overcome the above-mentioned challenges has promoted the development of synthetic receptor molecules that recognise and bind methyllysines. Such "artificial antibodies" are of interest for a number of applications, for example, as reagents in biochemical assays, for the isolation and purification of post-translationally methylated proteins and for the tracking of signalling pathways. Moreover, they offer new approaches in diagnostics and therapy. This review delivers an overview of the broad field of methyllysine binding and covers a wide range of synthetic receptors used for the recognition of methylated lysines, including calixarenes, resorcinarenes, pillararenes, disulfide cyclophanes, cucurbiturils and acyclic receptors.

Binding Modes of a Phenylpyridinium Styryl Fluorescent Dye with Cucurbiturils

“…The performance of a CB7-PhSt system as indicator displacement assay was tested with lysine (Lys), methyl lysine (LysMe), dimethyl lysine (LysMe 2 ), and trimethyl lysine (LysMe 3 ) as model analytes. Methods for the discrimination of lysine and methylated lysines are of key importance in biochemistry [47] since the methylation of the lysine residuals of histones is an important mechanism of epigenetic regulation of gene expression.…”

Achieving High Affinity and Selectivity for Asymmetric Dimethylarginine by Putting a Lid on a Box

“…These PTMs are of significant interest due to their role in regulating gene expression and their dysregulation in numerous diseases . Much work has gone into developing synthetic receptors that bind PTMs selectively in aqueous solution, with applications in sensing, tagging, enrichment, and enzymatic assays . However, achieving selectivity for one methylation state of Lys or Arg has been a persisting challenge, since all eight possible methylation states can participate in electrostatic interactions, cation–π interactions, and H‐bonding, with the exception of trimethyllysine (Kme3) which cannot H‐bond.…”

Structural basis for antigen recognition by methylated lysine–specific antibodies

“…For both ease-of-use and adaptability to currently available techniques, monoclonal antibodies remain ideal tools for the recognition of site-specific posttranslational modifications. While several studies have attempted to create artificial receptors that can recognize methylated lysines (12), these cannot be easily employed in basic immunochemical assays such as western blotting, immunocytochemistry, and immunohistochemistry due to low affinity and a lack of sequence specificity surrounding the modification site. Indeed, protein methylation is considered one of the most difficult targets to create modificationspecific antibodies due to the minute differences in chemical moieties brought by the addition of -CH3 group(s) (10).…”