The protein n16 is a framework protein associated with biogenic aragonite stabilisation

in nacre, known to play a critical role in three key interfaces in nacre; the protein/ß –chitin, protein–protein, and protein–aragonite interfaces. The conformations of n16N, the N-terminal fragment of n16 is thought to be active at all three interfaces¹ . We investigate n16N under aqueous conditions through the use of replica exchange with solute tempering (REST)²  molecular dynamics simulations. n16N has been previously proposed to be intrinsically disordered in character. Our key aim is to rigorously characterise this ensemble of conformations for n16N in aqueous solution, and explore current hypotheses regarding the possible sub-domain organisation of n16N, as related to these three key interfaces³ . We confirm that n16N supports an equilibrium ensemble featuring a wide range of conformations that lack well-defined secondary structure, providing further evidence for the suggested intrinsically-disordered character of this peptide. We have also highlighted the effect of the presence of Ca²⁺ ions on the structure of n16N, as identified from previous experimental studies¹ . Our simulations indicate that tyrosine plays a multi-functional role in stabilising conformations of n16N, for both intra-peptide and possibly inter-peptide interactions. Our findings reveal that the N-terminal half of the peptide can be structurally-stabilised via tyrosine–tyrosine interactions. In contrast, the C-terminal half of the peptide, lacking in tyrosine, shows substantial conformational lability, and in partnership with the highly-charged character of this sub-domain, appears a likely nucleation site for calcium carbonate. Finally, dominant structures from our predicted conformational ensemble of n16N suggest the outward presentation of residues thought to be critical to binding to ß-chitin⁴ . Our data supports the hypothesis relating to the presence of different functional sub-domains within n16N.