Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin

Table S1. Amidated cationic membrane-lytic and antimicrobial peptides. There are myriad cationic membrane-lytic and antimicrobial peptides consisting of a C-terminal amidation post-translational modification that are of high interest to researchers and which our recombinant expression followed by chemical amidation could be immediately applied to. Below is a short representative list of such peptides.

Table S2. Melittin backbone chemical shifts (ppm). Deuterium isotope shift corrections were applied to all chemical shifts and pressure corrections were applied to the pressure denatured monomer chemical shifts. All shifts were measured on a 1.0-mM ²H,¹³C,¹⁵N-labeled melittin sample in 25-mM potassium phosphate buffer, pH 7.0, 50-mM NaCl, 3% D2O. The folded tetramer peaks were measured at atmospheric pressure, while the pressure denatured monomer resonances were measured at 2.25 kbar. 

Table S3. Melittin experimental ¹⁵N-¹H isotropic J-couplings, (J+¹D_NH) anisotropic couplings and ¹D_NH residual dipolar couplings. All couplings and errors are reported in Hz. Isotropic J-couplings were not measured for G3 and A4. In these cases, an average J-coupling of -93.3 Hz was used with an uncertainty of 1.1 Hz..

Table S4. Analysis of the AlphaFold-Multimer structural models with respect to the melittin RDCs measured with the Pf1 alignment media. SVD fitting of the RDC alignment tensor parameters was done for residues 3-23 in the AF-M models and residues 3-25 in the 2MLT crystal structure. The AF-M model confidence score is a prediction of the similarity between the AF-M model and the true oligomeric structure. The RMSD listed in the final column is the backbone RMSD between the AF-M structural models and the 2MLT X-ray crystal structure tetramer for residues 1-23.

Table S5. Analysis of the AlphaFold-Multimer structural models with respect to the melittin RDCs measured with a stretched polyacrylamide gel alignment media. SVD fitting of the RDC alignment tensor parameters was done for residues 4-23 in the AF-M models and residues 4-25 in the 2MLT crystal structure. The AF-M model confidence score is a prediction of the similarity between the AF-M model and the true oligomeric structure. The RMSD listed in the final column is the backbone RMSD between the AF-M structural models and the 2MLT X-ray crystal structure tetramer for residues 1-23.

Table S6. RDC based ranking of melittin AlphaFold-Multimer structural models and the 2MLT crystal structure. Models are ranked according to the average Q factor in the two alignment media used in this study: Pf1 and a positively charged stretched polyacrylamide gel. The AF-M model confidence score is a prediction of the similarity between the AF-M model and the true oligomeric structure. The RMSD listed in the final column is the backbone RMSD between the AF-M structural models and the 2MLT X-ray crystal structure tetramer for residues 1-23.

 Table S7. 2MLT crystal structure and AlphaFold-Multimer atomic coordinates. The X-ray crystal structure was retrieved from the protein data bank (PDB), then it was symmetry expanded into a tetramer, residues were renumbered as described in 3.3.3 Comparison of ¹D_NH couplings with the crystal structure and AlphaFold-Multimer model, and finally protons were added to the structure with DYNAMO. The C-terminus column lists the C-terminal modifications for the AF-M structures in comparison to melittin’s native -CONH₂ C-terminus. 

Atomic coordinates in the form of .pdb files for all RDC SVD fitting done in this study: