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 (RH) of around 15–16 nm. Upon heating to 37°C, particles of P41T increased in size (RH = 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.

Keywords:
Enamel, Amelogenin, Amelogenesis imperfecta, Dynamic light scattering, Thermal denaturation, Fluorescence spectroscopy, Protein aggregation
1.
Barron, M.J., S.J. Brookes, J. Kirkham, R.C. Shore, C. Hunt, A. Mironov, N.J. Kingswell, J. Maycock, C.A. Shuttleworth, M.J. Dixon (2010) A mutation in the mouse Amelx tri-tyrosyl domain results in impaired secretion of amelogenin and phenocopies human X-linked amelogenesis imperfecta. Hum Mol Genet 19: 1230–1247.
2.
Collier, P.M., J.J. Sauk, J. Rosenbloom, Z.A. Yuan, C.W. Gibson (1997) An amelogenin gene defect associated with human X-linked amelogenesis imperfecta. Arch Oral Biol 42: 235–242.
3.
Fincham, A.G., J. Moradianoldak. P.E. Sarte (1994) Mass-spectrographic analysis of a porcine amelogenin identifies a single phosphorylated locus. Calcif Tissue Int 55: 398–400.
4.
Lakshminarayanan, R., K.M. Bromley, Y.P. Lei, M.L. Snead, J. Moradian-Oldak (2010) Perturbed amelogenin secondary structure leads to uncontrolled aggregation in amelogenesis imperfecta mutant proteins. J Biol Chem 285: 40593–40603.
5.
Lench, N.J., G.B. Winter (1995) Characterization Of molecular defects in X-Linked amelogenesis imperfecta (Aih1). Hum Mutat 5: 251–259.
6.
Moradian-Oldak, J. (2001) Amelogenins: assembly, processing and control of crystal morphology. Matrix Biol 20: 293–305.
7.
Moradian-Oldak, J., M.L. Paine, Y.P. Lei, A.G. Fincham, M.L. Snead (2000) Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy. J Struct Biol 131: 27–37.
8.
Paine, M. L., Y.P. Lei, K. Dickerson, M.L. Snead (2002) Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1). J Biol Chem 277: 17112–17116.
9.
Paine, M.L., M.L. Snead (1997) Protein interactions during assembly of the enamel organic extracellular matrix. J Bone Miner Res 12: 221–227.
10.
Simmer, J.P., A.G. Fincham (1995) Molecular mechanisms of dental enamel formation. Crit Rev Oral Biol Med 6: 84–108.
11.
Tanimoto, K., T. Le, L. Zhu, H.E. Witkowska, S. Robinson, S. Hall, P. Hwang, P. DenBesten, W. Li (2008) Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta. J Dent Res 87: 451–455.
12.
Uversky, V.N. (2002) What does it mean to be natively unfolded? Eur J Biochem 269: 2–12.
13.
Wright, J.T. (2006) The molecular etiologies and associated phenotypes of amelogenesis imperfecta. Am J Med Genet A 140A: 2547–2555.
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2011
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