Disulfide Bonds: used to prevent conformational changes between pre and post fusion transformation

• covalent linking a region that changes during the transition to an unchanging region, focusing on linking HR1 to either the central helix or the upstream helix.
• size-exclusion chromatography = larger than wild type
• negative stain electron microscopy = partially misfolded
• only 2 variants that linked the linked the same helix to different flexible loops against another protomer had a higher expression and greater thermostability.

Salt Bridges: used to neutralize charges within S protein

• increased expression and normal structure in trimeric spikes with two variants

Hydrophobic Residues: used to fill internal cavities within S protein

• hydrophobic substitutions in the fusion peptide between HR1 and the beta-hairpin
• increased expression in combination with salt bridges or proline subsitutions

Proline Residues: used to cap helices or hold loops in place in prefusion state of S protein

• increased expression and thermostability, structure supported by nsEM