ABSTRACT
Protein macrocyclization is a pivotal process in the stabilization of protein structures,
significantly enhancing their proteolytic stability and thermostability. While nature
elegantly accomplishes this through a diverse family of ligases, laboratory methods
typically rely on recombinant proteins engineered with unnatural amino acids and cysteine
crosslinkers. Herein, we present a biological metabolite 4-hydroxynonenal (4-HNE)
to selectively modify nucleophilic amino acids, cysteine (Cys), histidine (His), and
lysine (Lys) into electrophilic hemiacetals followed by cyclization via oxime chemistry.
This reaction demonstrates a broad substrate scope, enabling the modification and
cyclization of proteins with a wide range of three-dimensional structures and molecular
weights, from 5.8 to 60 kDa. The resulting cyclized proteins exhibit greater proteolytic
stability and enhanced thermal stability at elevated temperatures compared to their
uncyclized counterparts. This clearly underscores the critical role of cyclization
in preserving the intricate 3D structures of proteins and opens new avenues for advanced
protein engineering.
Key words
proteins - cyclization - chemoselectivity - biomimetic synthesis - bioorganic - chemistry
