Electrostatic interactions involving lysine make major contributions to collagen triple-helix stability.

Important stabilizing features for the collagen triple helix include the presence of Gly as every third residue, a high content of imino acids, and interchain hydrogen bonds. Host-guest peptides have been used previously to characterize triple-helix propensities of individual residues and Gly-X-Y triplets. Here, comparison of the thermal stabilities of host-guest peptides of the form (Gly-Pro-Hyp)3-Gly-X-Y-Gly-X'-Y'-(Gly-Pro-Hyp)3 extends the study to adjacent tripeptide sequences, to encompass the major classes of potential direct intramolecular interactions. Favorable hydrophobic interactions were observed, as well as stabilizing intrachain interactions between residues of opposite charge in the i and i + 3 positions. However, the greatest gain in triple-helix stability was achieved in the presence of Gly-Pro-Lys-Gly-Asp/Glu-Hyp sequences, leading to a T(m) value equal to that seen for a Gly-Pro-Hyp-Gly-Pro-Hyp sequence. This stabilization is seen for Lys but not for Arg and can be assigned to interchain ion pairs, as shown by molecular modeling. Computational analysis shows that Lys-Gly-Asp/Glu sequences are present at a frequency much greater than expected in collagen, suggesting this interaction is biologically important. These results add significantly to the understanding of which surface ion pairs can contribute to protein stability.