Project Summary Both naturally occurring and laboratory protein modifications are important fields of research since proteins are central to virtually every biological process. On one hand, protein post-translational modifications (PTMs) are essential for proper functioning of an organism and are associated with many diseases. It is of great significance to study PTMs in order to develop new therapeutic agents. On the other hand, continuing advances in noncanonical amino acid (ncAA) mutagenesis has provided powerful tools for the manipulation and study of protein function. This MIRA application seeks to merge investigations on both fronts of these research areas. The first general area of this MIRA project focuses on the co-translational modification of protein through genetic code engineering. The long-term goal is to explore innovative strategies for ncAA mutagenesis through reprogramming the genetic code. Our immediate and unique focus is on reprogramming codon language with quadruplet codons (Qcodons), although triplet nonsense codons are the predominant ones used in the field of genetic code expansion. Built on our pioneering efforts on tRNA engineering, we propose a new direction to improve Qcodon decoding efficiency through the identification and implementation of recoding signals. The use of recoding signals can also significantly mitigate a major concern over undesirable readthrough of endogenous stop codons with nonsense suppression-based noncanonical amino acid (ncAA) mutagenesis in live cell studies. We will also apply a Qcodon-dependent and ncAA-mediated control strategy to the development of HIV-1 vaccines, which represents a novel direction that was first demonstrated by my group. Such strategy will also be applied to the generation of vaccines against other pathogenic viruses or bacteria in the future. The second general area of this MIRA project is to investigate the role of protein tyrosine O-sulfation (PTS) in mammalian cell biology. Our initial efforts will focus on PTS of chemokine receptors as the first step of our long-term efforts to study the effects of PTS on receptor signaling in general. Furthermore, we seek to develop therapeutic agents targeting sulfated receptors. These two innovative projects have their own goals and significance, but are unified under our expertise in chemical/synthetic biology and are partially associated with each other. The long-term goal of my laboratory is not only to develop innovative and meaningful chemical/synthetic biology tools, but also to employ these tools to gain insights into biomedical processes for the development of novel therapeutics.