SITE-SPECIFIC PROTEIN FUNCTIONALIZATION USING ACTIVATED CYSTEINE BASED CHEMICAL BIOLOGY METHODS
Abstract
Proteins, as one of the most essential biomacromolecules, involve in various of cellular processes such as enzymatic metabolism, small molecule transportation, immune response, DNA replication, etc. within living organisms. To obtain enough biological tools that assist scientists to study the cellular functions of human proteins, protein functionalization, in the other word, the incorporation of novel functional groups onto protein sequences has become more and more important and well-developed.
Proteins containing a C-terminal modification are critical to the protein synthesis via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffers from premature hydrolysis and low compatibility with denatured conditions. To totally bypass the involvement of an enzyme for expressed protein ligation, we developed an activated-cysteine directed protein ligation (ACPL) technique using 2-nitro-5-thiocyanatobenzoic acid (NTCB) as cysteine cyanylating reagent for undergoing nucleophilic acyl substitution with amines including a number of L- and D-amino acids and hydrazine. The afforded protein hydrazides could be used further for expressed protein ligation. We demonstrated the versatility of this approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a C-terminal posttranslational modification, RNAse H that actively hydrolyzed RNA, and exenatide which is a commercial therapeutic peptide. The technique, which is exceedingly simple but highly useful, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible.
Dehydroalanine (Dha) exists natively in certain proteins and can also be chemically made from a protein cysteine. As a strong Michael acceptor, dehydroalanine in proteins has been explored to undergo reactions with different thiolate reagents for making close analogues of posttranslational modifications (PTMs) including a variety of lysine PTMs. We explored an NTCB-triggered dehydroalanine formation which is highly efficient when cysteine is at the flexible C-terminal end of the protein. We produced ubiquitin and ubiquitin-like proteins containing a C-terminal dehydroalanine residue with high yields. Although this method was found not effective when cysteine is at an internal region of a protein, we believe this method will find broad applications in studying ubiquitin and ubiquitin like protein pathways and functional annotation of many PTMs in proteins such as histones.
Citation
Qiao, Yuchen (2021). SITE-SPECIFIC PROTEIN FUNCTIONALIZATION USING ACTIVATED CYSTEINE BASED CHEMICAL BIOLOGY METHODS. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /195187.