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

Cells Tissues Organs. 2011;194(2-4):284-90. doi: 10.1159/000324342. Epub 2011 May 2.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amelogenin / chemistry*
  • Amelogenin / genetics*
  • Amelogenin / metabolism
  • Amelogenin / ultrastructure
  • Animals
  • Circular Dichroism
  • Fluorescence
  • Humans
  • Hydrogen-Ion Concentration
  • Mice
  • Mutant Proteins / chemistry*
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Point Mutation / genetics*
  • Protein Folding*
  • Protein Structure, Quaternary
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / ultrastructure
  • Temperature

Substances

  • Amelogenin
  • Mutant Proteins
  • Recombinant Proteins