Poster Presentation 11th Annual Conference of the International Chemical Biology Society 2022

Protein Semi-Synthesis via Expressed Protein Selenoesters (#144)

Sameer S. Kulkarni 1 2 , Emma E. Watson 1 , Joshua W. C. Maxwell 1 2 , Gerhard Niederacher 3 , Jason Johansen-Leete 1 2 , Sussane Huhmann 3 , Somnath Mukherjee 3 , Alexander R. Norman 1 2 , Julia Kriegesmann 3 , Christian F. W. Becker 3 , Richard J. Payne 1 2
  1. The University of Sydney, Sydney, NSW, Australia
  2. Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, Sydney, NSW, Australia
  3. Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, Vienna, Austria

While >70% of human proteins are functionalized with an array of post-translational modifications (PTMs) following biosynthesis on the ribosome,1 the effect of a specific PTM on the structure/activity of the vast majority of the proteome remains uncovered, due to the lack of tools to access this modified proteome in homogeneous form. Chemical protein synthesis has emerged as a powerful approach for generating site-specifically modified proteins. Particularly, the advent of native chemical ligation (NCL)2 has enabled construction of hundreds of modified proteins.3 However, synthetic access to proteins comprising >180 residues remains extremely challenging. To circumvent this size limitation, Muir and co-workers developed expressed protein ligation (EPL),4 a robust and highly efficient semi-synthetic variant of NCL. EPL relies on intein fusion constructs to generate corresponding protein thioesters through recombinant expression, which can be ligated with synthetic peptides bearing PTMs under an NCL manifold, furnishing large modified proteins in a single step.5 While EPL and NCL have revolutionized the field of protein science, these methods require milimolar concentrations of reacting fragments for the reaction to proceed meaning that these techniques cannot be used to generate poorly soluble or aggregation-prone proteins. To address these issues, we recently developed a new ligation technology called the diselenide-selenoester ligation (DSL).6 DSL involves chemoselective reaction between a peptide bearing an N-terminal selenocystine and a peptide selenoester. Remarkably, these reactions proceed at unprecedented time scales (1-10 min, even at sterically hindered junctions) without any exogenous additives.7 Importantly, the deselenization step can be performed chemoselectively in the presence of unprotected Cys residues, and reactions can be performed at µM-nM concentrations, providing a significant advantage over NCL-based methods. In this presentation, I will describe the development and application of a novel semi-synthetic methodology, dubbed the expressed protein selenoester ligation (EPSL). The power and utility of EPSL will be described through the efficient semi-synthesis of differentially phosphorylated variants of the oligomeric chaperone protein Hsp27 and lipidated analogues of the membrane-associated GTPase YPT6.8