Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in E. coli . Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and is thus a prerequisite for studying its native structure and function. Here, we present a method for producing triply isotopically labeled ( 2 H, 13 C, and 15 N) native melittin through recombinant expression followed by chemical amidation. We then show that structural models produced with AlphaFold-Multimer are in even better agreement with experimental residual dipolar couplings than the 2.0 Å resolution X-ray crystal structure for residues G3-K23.