Equimolar Cross-Coupling Using Reactive Coiled Coils for Covalent Protein Assemblies
The Pentelute Lab aims to invent new chemistry for the efficient and selective modification of proteins, to ‘hijack’ these biological machines for efficient drug delivery into cells and to create new machines to rapidly and efficiently manufacture peptides and proteins.
Pentelute Lab, Chemistry, MIT, Chemistry Department, Boston, Cambridge, Biology, Peptides, Peptide, Proteins, Science, Rapid, Brad Pentelute, Brad,
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Equimolar Cross-Coupling Using Reactive Coiled Coils for Covalent Protein Assemblies

Equimolar Cross-Coupling Using Reactive Coiled Coils for Covalent Protein Assemblies

Hironori Takeuchi Elee Shimshoni Satish Gandhesiri Andrei LoasBradley L. Pentelute*

Abstract

Biocompatible cross-coupling reactions enable the efficient covalent attachment of large biomolecules at near-stoichiometric ratios, ensuring the stability and integrity of the resulting products. We present an affinity-based peptide platform utilizing coiled coils containing reactive side chains for proximity-driven protein cross-coupling in the presence of a cross-linking agent. This platform supports both chemical synthesis and recombinant expression, using canonical amino acids to generate reactive affinity tags. Employing the E3/R3 coiled coil pair as a scaffold, we design four complementary coils with cysteine residues as cross-linking sites, achieving >90% conversion to covalent heterodimeric coupling products using 3,4-dibromomaleimide. Equimolar mixtures of proteins with reactive coils at their termini yield near-quantitative heterodimeric cross-coupling products. The strategic selection of complementary coiled coil pairs and cross-linking agents enables orthogonal assembly of macromolecules with diverse architectures. This method offers a versatile approach for creating covalent fusion proteins, enhancing their stability and functionality for applications in chemical biology, biotechnology, and medicine.

Category
2024, Publications