Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy

Dynamic light scattering (DLS) analysis together with atomic force microsco py (AFM) imaging was applied to investigate the supramolecular self-assembl y properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogeni n to protein-protein interactions during the self-assembly process. Mouse a melogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino ac id mutations identical to those found in cases of antelogenesis imperfecta were analyzed, The polyhistidine-containing full-length recombinant ameloge nin protein [rp(H)M180] generated nanospheres with monodisperse size distri bution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating t hat this histidine modification did not interfere with the self-assembly pr ocess. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG; epitope (''A'"-domain deletion) resulted in t he formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent d ynamic light scattering analysis of amelogenin molecules lacking amino acid s 157 through 173 and containing a hemagglutinin epitope ("B"-domain deleti on) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of large r and more heterogeneous nanospheres, The above data suggest that while the N-terminal-A-domain is involved in the molecular interactions for the form ation of nanospheres, the carboxy-terminal B-domain contributes to the stab ility and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolyti c cleavage of amelogenin at amino- and carboxy-terminii occurring during en amel formation influences amelogenin to amelogenin interactions during self -assembly and hence alters the structural organization of the developing en amel extracellular matrix, thus affecting enamel biomineralization. (C) 200 0 Academic Press.