Analysis of secondary structure and self-assembly of amelogenin by variable temperature circular dichroism and isothermal titration calorimetry

Abstract

Amelogenin is a proline-rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self-assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary-secondary structure and self-assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full-length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered beta-structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self-assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (DeltaH(A)) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions.

Fig. 1

Amino acid sequence of porcine amelogenin. The recombinant amelogenin used in this study lacks the first methionine and a phosphate group on serine16. The PXP/PXXP motifs that play an important role in imparting the PPII structure are highlighted in grey.

Fig. 2

(A) VT-CD spectra of rP172 in Tris-HCl buffer (pH = 7.4) containing 5 mM CaCl₂. The protein concentration used was 20 μM. The inset shows the iso-elliptic point around 211 nm. (B) Difference spectra obtained by subtracting the CD spectra at 5 °C and 10 °C from the CD spectra at 45 °C. (C) Changes in CD intensity at 224 nm as a function of temperature. (D) Thermal dependency of CD intensity at 224 nm as a function of temperature in PBS buffer (pH 7.4). The black open circles and grey open circles indicate heating and cooling cycles, respectively. Note that the ellipticity is linear over a broad temperature range.

Fig. 3

Concentration dependent conformational changes in rP172 (A) CD spectra of various concentrations of rP172 at 10 °C (A) and 40 °C (B). (C) The ellipticity values at 224 nm as a function of concentration at 10 °C (solid circles) and at 40 °C (open circles). Note that the β-structured assembly is rapidly reached at elevated temperature.

Fig. 4

ITC dilution studies of assembled rP172 at various temperatures. (A, B) Raw ITC data for the dilution of rP172 at 30 °C and 37 °C. The temperature is indicated in each enthalpogram. (C) Heat of dilution (δ_qi) per mole of rp172 injected (δ_ni) as a function of concentration of rP172 in the cell at 30 °C and 37 °C. (D) The molar heat of assembly as a function of temperature (based on three temperatures).

Fig. 5

Predicting the propensities for primary structures of rP172. (A) β-sheet or aggregation propensity (red) and (B) PPII propensity (blue) for rP172. Each point represents average propensity of 5 contiguous residues. The broken line in the figure indicates mean propensity of the whole protein. The regions higher than this line are the stretches that, when compared to the whole sequence, have high aggregation, or β-sheet, and PPII propensity. (C) Amino acid sequence of rP172 with the regions having high PPII (blue letters) and aggregation (red letters) propensities. The overlapping and unknown regions are labeled in green and black, respectively. The A- and B- domains that were shown to play an important role in the self-assembly and protein-protein interactions are indicated by the horizontal lines.

Fig. 6

Proposed modular structure for rP172 with alternate PPII and hydrophobic domains. The domains with high β-sheet propensity are involved in protein-protein interactions and aid the self-assembly whereas PPII domains provide water solubility and prevents excessive aggregation.