Intracellular bismuth coordination of peptides and miniproteins enables functional screening of stable cyclic architectures

Abstract Cyclisation of peptides and proteins is a powerful chemical strategy to enhance molecular stability and functional efficacy, yet executing coordination chemistry within living cells remains challenging. Bismuth(III) coordination represents a particularly compact and chemoselective cyclisation modality, yet to date has remained confined to in vitro or phage-based screening systems. Here we report bismuth(III)-thiolate coordination chemistry within living cells during recombinant expression in E. coli , enabling intracellular cyclisation of peptides and miniproteins. Supplementation of growth media with bismuth salts enabled efficient intracellular coordination of either three or six cysteine residues, yielding bicyclic and tetracyclic architectures with minimal cellular toxicity. Bis-bismuth coordination generated a tetracyclic miniprotein with enhanced thermal and serum stability, representing a level of intracellular structural reinforcement not previously accessible. To validate this chemistry in a functional selection format, we integrated intracellular bismuth coordination with an alpha-synuclein protein-fragment complementation assay, screening a genetically encoded library to enrich a bismuth-constrained bicyclic peptide that reduced α-synuclein aggregation. Intracellular bismuth coordination therefore enables peptide and miniprotein constraint during expression, preserving genotype–phenotype linkage and expanding the topological space accessible to live-cell screening platforms.