Lys2 has previously been implicated as a residue important in binding interactions between omega-conotoxins and the N-type calcium channel. To further assess the importance of this residue, Lys2 to Ala2 derivatives of omega-conotoxins MVIIA and CVID were synthesized and their structures and binding potencies determined. A comparison of the 3D structures of the Ala2 mutants with the parent peptides suggest there are significant structural differences brought about by this substitution. In particular, stabilizing interactions between Lys2 and loop 2 of the omega-conotoxins are removed, leading to greater flexibility in loop 2, which contains Tyr13, a crucial residue for omega-conotoxin binding to the N- and P/Q-type voltage-gated calcium channel (VGCC). The significant drop in binding potencies resulting from replacement of Lys2 thus appears to relate more to entropic factors than to any direct interaction of Lys2 with the VGCC. This has significant implications for the development of a pharmacophore binding model for omega-conotoxins, as removal of Lys2 from consideration suggests that the omega-conotoxins residues that interact with the N-type VGCC reside in loop 2 and 4, and thus cover a significantly smaller and more defined area of the surface of omega-conotoxin than previously thought.