Recent advances in protein modifications techniques for the targeting N‐terminal cysteine

Abstract

N-terminal modifications of proteins have garnered the attention of chemical biologists because of their critical roles in numerous cellular processes, including protein translocation, protein structural and metabolic stability, and the regulation of the half-life of proteins by a process known as "N-end rule pathway." In addition, other posttranslational modification processes also depend on the N-terminal signal sequences. Chemical probes are powerful tools that can be used to investigate N-terminal modifications, to gain structural and functional insights into protein modification, protein-protein interactions, protein localization, and protein dynamics. Most modifications target cysteine and lysine residues because of their high nucleophilicity. However, due to multiple occurrences of these residues in a protein at a given time, regioselective labeling can be quite difficult to accomplish. N-terminal cysteine presents itself as a unique practical option to overcome regioselectivity and site-specificity challenges. This review provides an overview of N-terminal cysteine labeling tools, including N-terminal cysteine condensation with aldehydes, cyanobenzothiazoles, cyclopropenones, and trans-thioesterification. The review also highlights the technical challenges of each of the techniques, which need to be addressed to broaden the scope of these approaches.

Conjugates of Recombinant Protein‐Based Polymers: Combining Precision with Chemical Diversity

“…However, since most globular proteins contain multiple instances of such AAs, this approach often results in off‐target labeling or a heterogeneous product with the attendant problems of impaired folding or functioning of the modified protein. To address this, genetic engineering methods can be utilized to incorporate a unique reactive AA provided this AA is not already present in the protein sequence or exposed to the accessible protein surface, [ 13 ] at a distinct (e.g., N‐terminal) positions, [ 14 ] or if other instances of this AA can be mutagenized without a significant loss of protein function. [ 6,15 ] Still, particularly for PBPs, for which sequence complexity is usually low, the identification or addition of a unique reactive AA is more probable, unless the fusion of another protein/s or peptide/s to the PBPs is desired (which can reintroduce AA heterogeneity).…”

Opportunities for Expanding Encoded Chemical Diversification and Improving Hit Enrichment in mRNA‐Displayed Peptide Libraries

“…[93] Using a solid support approach it was shown that aldehydes in particular can undergo an 'explosion' of diversity from one precursor into many different structures (including olefins, alkynes, amides, amines, β-hydroxyand β-aminoketones, and diverse heterocycles). [94] N-terminal cysteines also offer possibilities for further modification, [95] with native chemical ligation being an obvious example that can be used to attach diverse thioester precursors to the N-terminus. [96] Nitrile-based reagents also provide an option for clean N-terminal modification in proteins and should be compatible with mRNA display, for example cyanobenzothiazoles [97] (although some sequence-specific reactions with other cysteines are possible and cannot be neglected in a peptide library setting).…”

“…N ‐terminal cysteines also offer possibilities for further modification, [95] with native chemical ligation being an obvious example that can be used to attach diverse thioester precursors to the N ‐terminus. [96] Nitrile‐based reagents also provide an option for clean N ‐terminal modification in proteins and should be compatible with mRNA display, for example cyanobenzothiazoles [97] (although some sequence‐specific reactions with other cysteines are possible and cannot be neglected in a peptide library setting).…”

Fast and Selective Reaction of 2-Benzylacrylaldehyde with 1,2-Aminothiol for Stable N-Terminal Cysteine Modification and Peptide Cyclization

“…Reactions that are specific to N-terminal Cys residues have been used for site-specific protein labeling, protein semisynthesis, and peptide cyclization. − One reaction that has been widely used is the native chemical ligation (NCL), , which proceeds through transthioesterification of N-terminal Cys and subsequent S-to-N acyl exchange . Another reaction relying on selective thiazolidine formation between aldehyde and 1,2-aminothiol has also been widely used, which however takes long reaction times under acidic conditions .…”