Probing the self‐association, intermolecular contacts, and folding propensity of amelogenin

Abstract

Amelogenins are an intrinsically disordered protein family that plays a major role in the development of tooth enamel, one of the most highly mineralized materials in nature. Monomeric porcine amelogenin possesses random coil and residual secondary structures, but it is not known which sequence regions would be conformationally attractive to potential enamel matrix targets such as other amelogenins (self-assembly), other matrix proteins, cell surfaces, or biominerals. To address this further, we investigated recombinant porcine amelogenin (rP172) using ''solvent engineering'' techniques to simultaneously promote native-like structure and induce amelogenin oligomerization in a manner that allows identification of intermolecular contacts between amelogenin molecules. We discovered that in the presence of 2,2,2-trifluoroethanol (TFE) significant folding transitions and stabilization occurred primarily within the N-and C-termini, while the polyproline Type II central domain was largely resistant to conformational transitions. Seven Pro residues (P2, P127, P130, P139, P154, P157, P162) exhibited conformational response to TFE, and this indicates these Pro residues act as folding enhancers in rP172. The remaining Pro residues resisted TFE perturbations and thus act as conformational stabilizers. We also noted that TFE induced rP172 self-association via the formation of intermolecular contacts involving P4-H6, V19-P33, and E40-T58 regions of the N-terminus. Collectively, these results confirm that the Nand C-termini of amelogenin are conformationally responsive and represent potential interactive sites for amelogenin-target interactions during enamel matrix mineralization. Conversely, the Pro, Gln central domain is resistant to folding and this may have important functional significance for amelogenin.Abbreviations: AQ-rP172, uniformly labeled 13 C, 15 N rP172 in 90% v/v UDDW, 10% v/v D 2 O, pH 3.8; CD, circular dichroism; DLS, dynamic light scattering; HSQC, heteronuclear single quantum coherence; IDP, intrinsically disordered protein; rP172, recombinant porcine amelogenin; 70-rP172, uniformly labeled 13 C, 15 N rP172 in 30% v/v UDDW, 70% v/v 2,2,2-trifluoroethanol, pH 3.8; RC, random coil; SSP, secondary structure propensity score; TFE, 2,2,2-trifluoroethanol; UDDW, Milli-Q pure unbuffered deionized distilled water.

Amelogenin and enamel biomimetics

“…40 Based on the PPII propensity scale for individual amino acid residues, we previously reported that PPII is the dominant structure in the central region of amelogenin 33 and our solvent engineering studies confirmed that the Pro, Gln central domain is resistant to folding. 40 The rigidity in the central region and flexibility at the N- and C- termini may have important functional significance for amelogenin in vivo . 40 …”

“…40 The rigidity in the central region and flexibility at the N- and C- termini may have important functional significance for amelogenin in vivo . 40 …”

“…(A) Amelogenin monomer in acidic solution; Reproduced with permission from ref 27. (B) In 70% TFE; Reproduced with permission from ref 40. (C) In 10% SDS; Reproduced with permission from ref 41.…”

Intrinsically Disordered Proteins in Biomineralization

“…Mammals DMP1 4.0 CD, DLS, FTIR, SAXS (Gericke et al, 2010;He et al, 2003aHe et al, , 2003b DPP 2.8 CD, NMR, SAXS (Cross et al, 2005;Evans et al, 1994;Fujisawa & Kuboki, 1998;George & Hao, 2005;He et al, 2005b;Lee et al, 1977) BSP 4.1 CD, NMR, SAXS (Fisher et al, 2001;Tye et al, 2003Tye et al, , 2005Wuttke et al, 2001) OPN 4.4 CD, NMR, FTIR (Fisher et al, 2001;Gorski et al, 1995) amelogenin 6.6 CD, NMR (Buchko et al, 2010;Delak et al, 2009b;Ndao et al, 2011;Shaw et al, 2008) statherin 8.0 CD, NMR (Long et al, 2001;Naganagowda et al, 1998;Raj et al, 1992) lithostathine 5.7 CD (Gerbaud et al, 2000) Haliotis rufescens AP7 5.2 CD, NMR (Kim et al, 2004(Kim et al, , 2006aMichenfelder et al, 2003;Wustman et al, 2004) Atrina rigita Asprich 2.7-3.5 CD, NMR (Collino et al, 2006;Delak et al, 2009aDelak et al, , 2008Kim et al, 2008;Ndao et al, 2010) Procambrus clarkii CAP-1 3.9 CD (Inoue et al, 2007) Strongylocentrotus purpuratus SM50 10.8 CD, NMR (Xu & Evans, 1999;Zhang et al, 2000) PM27 8.1 CD, NMR Danio rerio Stm 4.1 CD, gel filtration (Kaplon et al, 2008(Kaplon et al, , 2009 Table 1. IDPs involved in biomineralization of calcium carbonate and phosphate for which a disordered structure has been confirmed...…”

“…There is no well-defined, continuous region with an ordered structure, but some residual secondary structure was observed. In addition, amelogenin exists in at least two conformations (Buchko et al, 2010;Delak et al, 2009b;Ndao et al, 2011). Amelogenin can interact with other important proteins engaged in enamel formation (Bartlett et al, 2006).…”