Dissecting neurofilament tail sequence-phosphorylation-structure relationships with multicomponent reconstituted protein brushes

Abstract

Neurofilaments (NFs) are multi-subunit, bottlebrush-shaped intermediate filaments abundant in the axonal cytoskeleton, with "bristles" composed of the subunits' disordered tail domains. Precisely how the tails' variable charge patterns and repetitive phosphorylation sites mediate their conformation within the brush remains an open question in axonal biology. We address this problem by grafting recombinant NF tail protein constructs (NFL, NFM, and NFH) to functionalized substrates, forming phosphorylatable brushes of defined stoichiometry. Atomic force microscopy reveals that NFM-based brushes are highly extended, while brushes incorporating the much larger NFH are surprisingly compact even after multisite phosphorylation. A self-consistent field theory predicts multilayered brush morphologies for NFM and phosphorylated NFH brushes. Further experiments with designed mutants reveal that N-terminal negative charges in NFH repel phosphorylated residues to generate the multilayer morphology and that charge segregation in NFM promotes collapsed conformations, lending new insight into how NF tail sequence features determine protein brush conformation.