Folding, assembly, and aggregation of recombinant murine amelogenins with T21I and P41T point mutations

Abstract

Two point mutations (T21I and P40T) within amelogenin have been identified from human DNA sequences in 2 instances of amelogenesis imperfecta. We studied the folding and self-assembly of recombinant amelogenin (rM180) compared to the T21I and P40T mutants analogs. At pH 5.8 and 25°C, rM180 and the P41T mutant existed as monomers, whereas the T21I mutant formed small oligomers. At pH 8 and 25°C, all of the amelogenin samples formed nanospheres with hydrodynamic radii (R(H)) of around 15-16 nm. Upon heating to 37°C, particles of P41T increased in size (R(H) = 18 nm). During thermal denaturation at pH 5.8, both of the mutant proteins refolded more slowly than the wild-type (WT) rM180. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that the WT transformed to a partially folded conformation upon heating and remained stable. Thermal denaturation and refolding studies indicated that the mutants were less stable and exhibit a greater ability to prematurely aggregate compared to the WT. Our data suggest that in the case of P41T, alterations in the self-assembly of amelogenin are a consequence of destabilization of the secondary structure, while in the case of T21I they are a consequence of change in the overall hydrophobicity at the N-terminal region. We propose that alterations in the assembly (i.e. premature aggregation) of mutant amelogenins may have a profound effect on intra- and extracellular processes such as amelogenin secretion, proteolysis, and its interactions with nonamelogenins as well as with the forming mineral.

Fig. 1.

TEM images showing the T21I mutant at pH 5.8, 0.4 mg/ml, and 37°C (a). The particles were 18.2 nm in diameter on average, which is in approximate agreement with DLS. White arrows highlight 5 of the most clearly apparent oligomers. b The P41T mutant formed large aggregates at 37°C with diameters ranging from 100 to 500 nm, again in agreement with DLS. Scale bars = 100 and 500 nm, respectively.

Fig. 2.

First heating (solid lines) and cooling (dashed lines) CD profiles performed at pH 5.8 (monitoring at 224 nm) of rM180 (a), T21I (b), and P41T (c) measured at pH 5.8. Third heating (solid lines) and cooling (dashed lines) profiles of rM180 (d), T21I (e), and P41T (f).

Fig. 3.

a Fluorescence heating profiles of rM180, T21I, and P41T measured at pH 5.8. The maximum emission wavelength was plotted as the samples were heated. b VT light scattering plots of rM180, T21I, and P41T. The inset provides a close-up view of the area identified by the box, giving a clearer view of the onset of aggregation of T21I and P41T.