Modeling the Effect of Side-Chain Length on the Folding of Lactam-Modified Conotoxins

Abstract

We developed a method to model the folding of α-conotoxins by combining molecular dynamics (to determine geometric factors relevant to folding) with ab initio calculations (for molecular orbital information). In previous work, [Kovacs et al. Biophys. J. 2010, 98, 636a], we followed literature suggestions to direct specific regioisomer formation by replacing the Cys3 with aspartate and the Cys13 thiol with an ammonium (-NH3+) group. The results were encouraging because we observed directionality towards the proper regioisomer folding, but we hypothesized that the short side chains at positions 3 and 13 resulted in brief contact times between the carboxyl and amino groups and no C-N overlap. Now, we evaluate this hypothesis’ validity by lengthening the side chain at position 13. We carried out equivalent calculations with two (vs one), three and four methylene units (-CH2-) between the alpha carbon and the ammonium group (four methylene units make this amino acid a lysine). The results show that thermal fluctuations lead to configurations where a molecular orbital overlap between S-S atoms (Cys2-Cys7) can take place, leading to the proper regioisomer formation. Furthermore, the amino acid proline appears to generate rigidity in its surrounding amino acids, specifically in at least the region controlling the relative orientation of the Cys2 and Cys7 residues. The length of the methylene chain of the basic amino acid at position 13 affects the probability of forming a lactam bridge between positions 3 and 13. With short chains (one methylene group), there never is any observed orbital overlap between the carbon and nitrogen atoms, possibly because of the rigidity of the backbone. The probability of robust overlap increases with chain length and matches the efficiency of the Cys2-Cys7 overlap when using lysine at position 13.