Molecular Conformation of Porcine Amelogenin in Solution: Three Folding Units at the N-terminal, Central, and C-Terminal Regions1

Abstract

Circular dichroism (CD) studies were conducted to gain a better insight into the conformation of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90 degrees C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25 degrees C. It was tentatively concluded that the N-terminal region contains beta-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined.

Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide

“…In an overlapped region to this, peaks observed between 1,610 and 1,640 cm −1 were attributed to β-sheet (Susi and Byler, 1983; Jackson and Mantsch, 1995). Random coil conformation was attributed to peaks between 1,640 and 1,650 cm −1 (Krimm and Bandekar, 1986; Barth and Zscherp, 2002; Elangovan et al, 2007), which have also been reported in amelogenin (Renugopalakrishnan et al, 1986; Goto et al, 1993; Matsushima et al, 1998; Elangovan et al, 2007; Yang et al, 2010). Also, peaks observed between 1,650 and 1,655 cm −1 were attributed to α-helix conformation (Susi and Byler, 1983; Surewicz et al, 1993; Roach et al, 2005).…”

“…Peaks observed at 1,620–1,630 cm −1 were identified as hydrated PPII helix (Johnston and Krimm, 1971; Wellner et al, 1996; Elangovan et al, 2007). Earlier reports indicate that the full length amelogenins contain a significant PPII fraction (Renugopalakrishnan et al, 1986; Goto et al, 1993; Sogah et al, 1994; Lakshminarayanan et al, 2007, 2009). In an overlapped region to this, peaks observed between 1,610 and 1,640 cm −1 were attributed to β-sheet (Susi and Byler, 1983; Jackson and Mantsch, 1995).…”

Amelogenin Self-Assembly and the Role of the Proline Located within the Carboxyl-Teleopeptide

“…Following the initial description of amelogenin proteins in the early 1960s, scientists have attempted to gain information about its secondary and tertiary structure. Amelogenin circular dichroism spectrum suggests that the amelogenin N-terminus contains β-sheet structure, while the central region and C-terminal region exhibit a random-coil conformation [12]. It has been suggested that amelogenin protein fails to retain stable secondary or tertiary structure [13,14]; however, stable supermolecular structures (nanosphere) are observed [3].…”

Amelogenin Supra-Molecular Assembly in vitro Compared with the Architecture of the Forming Enamel Matrix

“…Several attempts have been made to obtain secondary and tertiary structural information for amelogenin. These included poorly resolved X-ray diffraction images of a developing bovine tooth enamel matrix, possibly showing a ␤ -or a cross-␤ -structure [Bonar et al, 1965]; circular dichroism and nuclear magnetic resonance (NMR) studies have led to suggestions that the molecule contains both ␤ -structures and ␤ -turns [Renugopalakrishnan et al, 1989;Goto et al 1993], but other work has indicated a highly mobile structure [Termine et al, 1979].…”

“…Its length can theoretically vary between 1 and 2 nm (assuming an ␣ -helix or a ␤ -strand conformation). The circular dichroism study has suggested that the C terminus of amelogenin is in a random coil conformation [Goto et al, 1993]. Modeled as a hard sphere for the whole molecule, the apparent radius would be 2.25-2.75 nm.…”