Manipulating Cation- Interactions of Reader Proteins in Living Cells with Genetic Code Expansion

Despite tremendous success in understanding the chemicalnatureand the importance of cation-& pi; interactions in a rangeof biological processes, particularly in epigenetic regulation, thedesign and synthesis of stronger cation-& pi; interactionsin living cells remain largely elusive. Here, we design several electron-richTrp derivatives and incorporate them into histone methylation readerdomains to enhance the affinity of the reader domains for histonemethylation marks via cation-& pi; interactions in livingcells. We show that this site-specific Trp replacement strategy isgenerally applicable for the engineering of high-affinity reader domainsfor the major histone H3 trimethylation marks, such as H3K4me3, H3K9me3,H3K27me3, and H3K36me3, with high specificity. Furthermore, we demonstratethat engineered reader domains can serve as powerful tools for theenrichment and imaging of histone methylation, as well as for capturingthe protein interactome at chromatin marks in living cells. Therefore,our study paves the way for the design of enhanced cation-& pi;interactions in reader proteins in living cells for various biologicalapplications.