Several conserved residues in the N-terminal helix have solvent-exposed side chains.
The side chains of several other highly conserved residues in the N-terminal helix project into solvent (Figure 8 ), including three in the vicinity of Trp120: Met118, Asp119, and Lys122 (corresponding to Met35, Asp36, and Arg39 in Ncb5or). Particularly notable in this group is Met118 (Met35). The thioether side chain of methionine is considered nonpolar, and indeed methionine is often subject to evolutionarily conservative replacement by residues with aliphatic side chains via single codon mutations (leucine, isoleucine, and valine). Only a few examples of such a mutation, specifically Met to Leu, exist among known Ncb5or proteins, and only in non-mammalian organisms (Figure S1 ). Methionine differs from the aliphatic amino acids it often replaces in that its thioether functional group can serve functional roles (for example, as a ligand to heme iron in cytochrome c ). The highly conserved nature of Met35 in Ncb5or, considered in the context of its solvent-exposed location, is suggestive of an essential functional role.