title: 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.
authors.affiliation: Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA.
authors.forename: Keith M
authors.initials: KM
authors.lastname: Bromley
authors.forename: Rajamani
authors.initials: R
authors.lastname: Lakshminarayanan
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Lei
authors.forename: Malcolm L
authors.initials: ML
authors.lastname: Snead
authors.forename: Janet
authors.initials: J
authors.lastname: Moradian-Oldak
mesh_terms: D053523:Amelogenin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D013696:Temperature
chemical_list: D053523:Amelogenin; D050505:Mutant Proteins; D011994:Recombinant Proteins