Post translational modifications in spider major ampullate silk influence structural conformation and N-terminal dimerization dynamics

Our current understanding of spider silk’s structure-function is largely based upon conclusions drawn from the comparison of amino acid sequences generated by genomics to techniques like NMR and X-ray crystallography. However, this comparison is ultimately limited to the extent that any given silk genome closely matches its proteome; with post-translational modifications (PTM) significantly influencing the structure and function of many proteins. This study utilizes phosphopeptide enrichment and mass spectrometry to produce a comprehensive PTM map across the major MaSp proteins of Trichonephila clavata, with PTMs observed in all structural domains. Solid-state NMR further confirms the presence and conformation of hydroxyproline, phosphoserine, phosphotyrosine and dityrosine, revealing phosphoserine’s roll in β-sheet inhibition. Moreover, N-terminal domain (NTD) phosphomimics where utilized to examine the impact of phosphorylation at the dimer interface, indicating that changes in charged clusters of the NTD monomers alters dimerization behavior in a manner that can possibly facilitate heterodimer formation.