A Modular Genetic Code Expansion Approach to Site-Specific Lysine Acylations

Lysine acylations are a diverse class of post-translational modifications (PTMs) that dynamically regulate protein function. Access to homogenous, site-specifically modified proteins is essential for dissecting their molecular roles, yet remains challenging with traditional methods. Here we present a modular strategy that combines genetic code expansion (GCE) with a chemoselective amide bond-forming reaction to install a wide range of lysine acylations directly onto folded proteins. Key to this approach is the genetic encoding of N ^ε -methoxylysine, which reacts efficiently with N -methyliminodiacetyl (MIDA) acylboronates to generate diverse acyl lysine modifications from a single protein precursor, including several acyl PTMs not previously accessible through GCE. This mild and broadly applicable methodology enables systematic studies of lysine acylation on multiple target proteins. We demonstrate its utility by probing the effects of defined acyl modifications on enzymatic activity, protein-RNA interactions and deacylase-mediated regulation. Together, this platform establishes a versatile strategy to interrogate the functional consequences of this wide-spread class of PTMs.