Development of Convergent Hybrid Phase Ligation for Chemical Synthesis of Challenging Proteins

Download or read book Development of Convergent Hybrid Phase Ligation for Chemical Synthesis of Challenging Proteins written by Ziyong Hong (Ph. D. in chemistry) and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Chemical protein synthesis has proven to be an important tool for investigation of protein structure and function, but application to large proteins remains a major challenge. Here we develop convergent hybrid phase native chemical ligation (CHP-NCL) as a new method for practical preparation of large proteins from multiple small, synthetically accessible peptide segments. This strategy combines the best features of ligation on the solid phase, to obtain reactive peptide “blocks” approximately 100 residues in length from several short peptide segments, and concepts from convergent ligation, which bring these blocks together for further ligation steps in solution phase. The parallel preparation of each block greatly improves the efficiency and overall yield of protein synthesis. We further carefully investigate the possible side reactions observed in the use of various derivatives of 3,4-aminobenzoic acid (Dbz), an enabling chemical functional group for this strategy, and provide guidance for the selection and use of appropriate variants in the preparation of activated peptide N-acylureas and thioesters. We demonstrate the feasibility of our CHP-NCL strategy on the synthesis of the 212-residue linker histone H1.2, with several combinations of post-translational modifications (PTMs) including citrullination, phosphorylation, and acetylation at sites that are hypothesized to play important roles in the regulation of chromatin structure and dynamics. We provide preliminary biochemical and biophysical characterization of the effect of these modifications on the interaction of H1.2 with nucleosomes. We anticipate that this new approach will find wide applications in the preparation of large proteins and facilitate the understanding of protein structure and function.