Year: 2026
Type: article
Abstract: ABSTRACT Modification of lysine residues is a common strategy in protein engineering, whether to prevent posttranslational modifications, control bioconjugation, or improve crystallization. The standard genetic approach—replacement with arginine by site‐directed mutagenesis—preserves positive charge but alters other physicochemical attributes and cannot address the N‐terminal amino group. Here, we characterize reductive methylation as a chemical alternative. This reaction converts every primary amino group to a dimethylamino group rapidly under mild aqueous conditions. Using human ribonuclease 1 and a cytotoxic variant engineered to evade the endogenous ribonuclease inhibitor as model systems, we assess the effects of complete dimethylation on thermostability, enzymatic catalysis, protein–protein interaction, compatibility with bioconjugation, cellular uptake, and intracellular persistence. Dimethylation preserves thermostability and a protein–protein interaction. Enzymatic catalysis, in contrast, is reduced by 10 2 ‐ to 10 3 ‐fold, consistent with the role of catalytic lysine residues. Dimethylation is fully compatible with bioconjugation chemistry. Dimethylated and unmodified ribonucleases show comparable uptake and persistence in human cells. These findings establish reductive methylation as a practical and conservative strategy for lysine modification in protein and peptide engineering and support its use in applications such as biological proteolysis‐targeting chimeras (bioPROTACs).
Source: Journal of Peptide Science
DOI: 10.1002/psc.70110
Institution: Massachusetts Institute of Technology
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,030
Funders: National Science Foundation, University of Wisconsin-Madison, Strong, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, National Institutes of Health, U.S. Air Force, National Defense Science and Engineering Graduate, National Cancer Institute, National Institute of General Medical Sciences, Air Force Research Laboratory
OpenAlex ID: W7164895642