Understanding the Role of Lysines and Phosphate Groups in PL12 Silicifying Peptide on Biomimetic Silica Formation in Marine Diatom T. pseudonana using Solid State NMR Measurements

Diatoms, unicellular algae abundant both in fresh & sea water, are famous for their beautifully organized nano & micro biosilica cell wall structures. In recent years, it was found that at the heart of biosilicification in diatoms, are the silaffin proteins rich in lysine (K) and phosphoserine groups. Investigation of silaffin 3 sequence from T. pseudonana diatom, revealed a repeating pattern of KxxKxxKxKxxK having a high affinity to biosilica ¹{Poulsen, 2013, Pentalysine Clusters Mediate Silica Targeting of Silaffins in Thalassiosira pseudonana}. How silaffins’ unique sequence is utilized to construct the intricate architecture of diatom cell walls is still unknown, though this question was extensively investigated^{2, 3} . We used PL12 (KAAKLFKPKASK), which is the shortest peptide from silaffin 3 that accelerates biomimetic silicification⁴ , to examine peptide regulation of silica morphology. Using NMR analysis, we compared between the secondary structure of the free peptide and peptide bound to biosilica. 2D ¹H-²⁹Si heteronuclear measurements showed correlation between Hε side chain of lysine & siloxane groups on the biosilica surface , giving direct evidence of lysine interaction with the less polar groups on the surface.

NMR measurements also showed that the five lysines are split into two groups: group A with 3 lysines in positions 1, 4, 12 and group B with lysines in positions 7, 9. Here, we investigate the role of the two groups & how they influence the precipitated biosilica structure. Two mutants were synthesized with the lysines replaced by alanines AAAALFKPKASA, KAAKLFAPAASK & then biomimetic silica was polymerized in their presence. We demonstrate the marked effect of the mutations on the silica structures formed & changes in the interaction of the lysine sidechain with the surface species between KAAKLFAPAASK & PL12.

To examine the role of phosphate groups in the silicification process², ³¹P measurements were done on biosilica in the presence of peptides. two speaks were found: free phosphate and a peak of phosphate bound covalently to the silicon via oxygen atom.