Post-transcriptional control of gene expression is critical for normal cellular function and viability. Poly(A)-binding protein (PABP) 1 is the prototypical member of a family of RNA-binding proteins which are key post-transcriptional regulators. PABP1 is multifunctional, acting as a primary determinant of translation efficiency and mRNA stability, regulating the fate of specific mRNAs, and participating in microRNAmediated regulation and nonsense-mediated mRNA decay. As well as binding various mRNAs, PABP1 achieves its multifunctionality through protein-protein interactions with numerous PABP-interacting motif (PAM)-2 motif-containing protein partners. These have been identified to bind the same site within the C-terminal PABC domain, therefore it is unclear how different PABP1 functions are coordinated. Recently, PABP1 was found to exhibit extensive post-translational modification (PTM), including putative lysine acetylation/methylation switches, which were suggested as a potential mechanism by which interactions with different PAM2 motifcontaining proteins may be regulated. In particular, in silico molecular modelling of the acetylation or dimethylation of the position 606 lysine residue (Lys606) within the PABC domain, using available structures of PABC in complex with PAM2 peptides of eukaryotic release factor (eRF)-3a and PABP-interacting protein (Paip)-2, suggested that modification of this residue, which is critical in PABC-PAM2 interactions, may differentially affect these PABP1 interactions. To examine the role of the Lys606 modification as a molecular switch to dictate PABC-mediated protein-protein interactions, site-specifically acetylated recombinant PABC domain was generated using cutting–edge amber codon suppression recoding technology. Following sequential purification by affinity, ion exchange and size exclusion chromatography, recombinant PABC protein quality was analysed by biophysical approaches such as thermal denaturation assay (TDA), dynamic light scattering (DLS), circular dichroism (CD) and liquid chromatography mass spectrometry (LCMS). Biochemical and biophysical analysis of PABC-PAM2 interactions was subsequently undertaken using GST-pulldown analysis, with the well characterised Paip2 protein, and Surface Plasmon Resonance (SPR) using PAM2 peptides of eRF3, Paip2 and trinucleotide repeat-containing (Tnrc) 6C (or GW182) proteins. These revealed that PABC Lys606 acetylation significantly increased the affinity and increased the association rate for eRF3 peptide. In contrast, effects on Paip2 peptide binding were less suggestive. Furthermore, although approaches to decipher the biological relevance of Lys606 and its modifications within cells are in their infancy, they reflect the complexity of studying PTM function in vitro. Overall, these data provide support for the hypothesis that Lys606 modification status confers selectivity between PABP1 protein partners suggesting a potential mechanism for how its multi-functionality may be coordinated.