This proposal is a competing renewal application of R01 GM117675 that uses a multidisciplinary approach to enhance our understanding of protein α-N-terminal methylation. The α-N-terminal methylation plays an essential role in regulating cell mitosis, chromatin interactions, and DNA repair. Its level is increased as response of cellular stress, aging, and developmental processes. The increasing occurrences of α-N-terminal methylation on the canonical X-P-K/R motif and noncanonical motifs highlight the importance of this underexplored modification. However, there are major gaps remain in our understanding of these fundamental biological processes. Filling these gaps is essential to elucidate the α-N-terminal methylation-mediated pathways and to enhance the opportunity to devise novel therapeutic approaches. The objective of this research is to develop novel chemical tools and apply them to understand the pathway and functions mediated protein α-N-terminal methylation. Meanwhile, we will elucidate the molecular basis for substrate specificity of human methyltransferase like 13 that methylates the the eukaryotic elongation factor 1 alpha containing the new GKEK motif at the α-N-terminus. Taken together, we believe that this research effort has the great potential to provide a clearer understanding of mechanisms and inhibition of NTMTs, and shed lights on the biological impact of protein α-N-terminal methylation. Accomplishment of the proposed work will provide new chemical tools for both basic biology research and has the opportunity to enhance the development of novel therapeutic approaches to target α-N-terminal methylation-involved pathways.