Effect of pH

Among the various environmental variables, the pH of the biomineralization microenvironment is a particularly important one, as BPPs tend to be highly charged. With 110 phosphoserine residues that account for about 50% of its amino acids^{Reference Anton, Nau and Nys26} and numerous aspartate and glutamate residues^{Reference Allerton and Perlmann32} phosvitin is an extraordinarily acidic protein. The pH-titration curve of phosvitin shows two equivalence points,^{Reference Mecham and Olcott25,Reference Giancotti, Quadrifoglio and Crescenzi33} with pK ₁ and pK ₂ values of approximately 7.2 and 2.5.^{Reference Taborsky34}

CD spectra recorded at different pH values show that these transitions are concomitant with a change in secondary structure (Figure 2).^{Reference Taborsky34,Reference Timasheff, Townend and Perlmann35} At pH 6.6, the phosvitin spectrum exhibits a strong negative band at 198 nm, combined with a weak positive and a weak negative band at 219 nm and 232 nm, respectively. Such a band pattern is in perfect agreement with an unordered conformation. Lowering the pH to 3.4 does not lead to any substantial spectral changes, except for a lower intensity of the shoulder at 220 nm. As stated previously, a shoulder at 220 nm indicates the presence of a P_II-helix. The P_II-helix has originally been described as the left-handed helical form adopted by oligo-l-prolines in water.^{Reference Rabanal, Ludevid, Pons and Giralt36,Reference Wilhelm, Lewandowski, Trapp and Wennemers37} However, this structural motif is present in a large number or even most unordered proteins and peptides and does not require the presence of a proline ring.^{Reference Sreerama and Woody16–Reference Toniolo, Formaggio, Woody, Berova, Polavarapu, Nakanishi and Woody18,Reference Tiffany and Krimm38,Reference Shi, Woody and Kallenbach39} Therefore, the pH-dependent change in the intensity of the shoulder at 220 nm in the CD spectrum of phosvitin indicates a transition between two different unordered conformations around physiological pH. It is a well-known phenomenon that secondary structure may change due to removal of electrostatic repulsion through pH-dependent neutralization of charged residues, allowing for a more compact, higher-order conformation.^{Reference Blout and Idelson40} In agreement with this, the spectrum at pH 1.8 shows characteristic features (maximum at ∼195 nm and minimum at ∼220 nm) of a β-sheet.

Figure 2.

Circular dichroism spectra of phosvitin recorded at pH 6.6, pH 3.4, and pH 1.8. Phosvitin (PV) has unordered conformation in a broad pH range and adopts a β-sheet structure at low pH. pH 6.6 adapted with permission from Reference 35. pH 3.4 and pH 1.8 adapted with permission from Reference 34.

Effect of interaction with cations

As the neutralization of negatively charged residues leads to conformational changes, a similar effect can be observed for phosvitin in the presence of cations. Effects of ionic strength^{Reference Grizzuti and Perlmann41} or Fe³⁺ ions^{Reference Taborsky42} on phosvitin structures have been reported. In the context of biomineralization, the effects of the bivalent cations Ca²⁺ and Mg²⁺ are of particular interest. For different pH, conformational transitions of phosvitin were concomitant with changes in the CD intensity at 220 nm (see Figure 2). Interestingly, the addition of Ca²⁺ ions generates a negative shoulder between 220 nm and 230 nm in the CD spectrum, which indicates β-sheet formation. On the other hand, the addition of Mg²⁺ ions results in a positive CD signal around 220 nm,^{Reference Grizzuti and Perlmann43} a hallmark for unordered structure containing the P_II-helix (Figure 3a).^{Reference Sreerama and Woody16–Reference Toniolo, Formaggio, Woody, Berova, Polavarapu, Nakanishi and Woody18} The presence of either Ca²⁺ or Mg²⁺ has an opposite effect on phosvitin conformation and suggests that the cation effect does not depend on charge neutralization alone. Not all charged groups are equally accessible for different cations. It has been assumed that this different selectivity for Ca²⁺ or Mg²⁺ leads to different conformations of the phosvitin/cation complex.^{Reference Grizzuti and Perlmann43}

Figure 3.

(a) Binding of bivalent cations to phosvitin (PV). (b) Ovocleidin-17 (OC-17) with and without Ca²⁺. (c) Interaction of PV with poly-l-lysine and protamine. Interaction with positive charge invokes conformational changes in avian biomineralization promoter proteins. (a) Adapted with permission from Reference 43. (b) Adapted with permission from Reference 44. (c) Adapted with permission from Reference 47.

The eggshell protein ovocleidin-17 also exhibits sensitivity toward addition of Ca²⁺ ions that is reflected by changes in the CD spectrum (Figure 3b).^{Reference Lakshminarayanan, Joseph, Kini and Valiyaveettil44} In absence of Ca²⁺, there is a minimum at 207 nm and a maximum below 195 nm that indicate the existence of domains with α-helical structure being in agreement with the crystal structure of ovocleidin-17.^{Reference Reyes-Grajeda, Jáuregui-Zúñiga, Rodríguez-Romero, Hernández-Santoyo, Bolanos-Garcia and Moreno45} However, because the α-helix spectrum has a much higher intensity than the spectra of other conformations, a CD spectrum might show the qualitative features of an α-helix, although it contains a significant amount of other secondary structures.^{Reference Woody, Berova, Polavarapu, Nakanishi and Woody9} Indeed, the existence of a shoulder around 196 nm in the CD spectrum of ovocleidin-17 also indicates the presence of unordered conformation.^{Reference Lakshminarayanan, Joseph, Kini and Valiyaveettil44} The addition of Ca²⁺ ions changes the curve shape and leads to a negative shoulder around 225 nm, which suggests some degree of β-sheet formation upon calcium binding. This would agree with the effect of Ca²⁺ binding to phosvitin, where the changes in the CD spectrum are consistent with a Ca²⁺-induced conformational change (Figure 3a).^{Reference Grizzuti and Perlmann43}

Charge effects are not restricted to the influence of pH and the presence of inorganic cations. Biomineralization occurs at organic matrices that are very often charged. Given that the pK values of the lysine and arginine side chains are 10.67 and 12.10, respectively,^{Reference Lide46} the polybases poly-l-lysine and arginine-rich protamine are generally charged at physiological (around neutral) pH and are therefore good models for organic polycationic materials. In the presence of poly-l-lysine or protamine, the CD spectrum of phosvitin has a β-sheet like appearance (Figure 3c).^{Reference Perlmann and Grizzuti47} As both poly-l-lysine and protamine have unordered structure, this indicates a conformational change in phosvitin invoked by the presence of a polycation. Compared to the β-sheet spectrum obtained at low pH (Figure 2) in the CD spectrum of the complex of phosvitin and poly-l-lysine, the minimum is considerably broader, and both the maximum and minimum are blue shifted. As mixing of phosvitin with polycations leads to cloudiness of the sample solution,^{Reference Perlmann and Grizzuti47} there may also be spectral contributions from differential scattering arising from aggregated particles of unordered conformation, as demonstrated in Figure 1b.

Effect of interaction with cations

EP fluid protein is a glycoprotein that has been purified from EP extracts of the blue mussel (Mytilus edulis).^{Reference Hattan, Laue and Chasteen53} It contains an unusual pattern of histidine (His) and aspartate (Asp) amino acid residues that occur in pairs: Asp-Asp-His-His-Asp-Asp-His-His-Asp···His-His-Asp. The overall high content of acidic residues (25.15%) suggests a role as a BPP. The CD spectrum of the EP fluid protein recorded in absence of calcium clearly indicates unordered conformation (Figure 4a).^{Reference Hattan, Laue and Chasteen53} The CD spectrum in the presence of Ca²⁺ has been assigned to α-helical conformation, however electrophoresis and sedimentation analyses have also shown that the EP fluid protein forms protomeric assemblies in the presence of calcium.^{Reference Hattan, Laue and Chasteen53} Therefore, similar to phosvitin in presence of polycations (Figure 3c), the CD spectrum may as well contain contributions from differential scattering (Figure 1b). This interpretation would allow a similar assignment of secondary structure as in Figure 3c and suggest that BPPs share structural properties across completely unrelated species.

Figure 4.

(a) Extrapallial (EP) fluid protein from Mytilus edulis in the absence and presence of Ca²⁺, which induces aggregation. (b) PfN44 in absence or presence of Ca²⁺ or Mg²⁺. A more pronounced spectral shift indicates a stronger affinity to Mg²⁺ than to Ca²⁺. (c) Different concentrations of N- and C-terminal peptides from nacre-associated protein n16. Spectral changes at higher concentration indicate associated protein/protein interactions. (a) Adapted with permission from Reference 53. (b) Adapted with permission from Reference 54. (c) Adapted with permission from Reference 61.

CD studies on phosvitin have shown that the effects of Ca²⁺ or Mg²⁺ on secondary structure may be different (see previous text). As the insertion of Mg²⁺ determines which polymorph of calcium carbonate is formed in mollusk shells, this property may play an important role in mineralization processes. PfN44, a protein from the Japanese pearl oyster Pinctada fucata, reduces the Mg²⁺-induced formation of aragonite, which leads to calcite crystals with significantly increased content of Mg²⁺ ions.^{Reference Pan, Fang, Xu, Liang, Zhang, Wang, Xie and Zhang54} The CD spectra recorded for PfN44 exhibit a minimum at ∼210 nm and a negative shoulder between 220 and 230 nm, which would agree with the dispersive curve shape of a spectrum that represents a predominantly unordered protein, even in the absence of bivalent cations (Figure 4b). Upon addition of Ca²⁺ or Mg²⁺, the minimum at 210 nm becomes less intense. In the presence of Mg²⁺, this intensity decrease is concomitant with an increase of the shoulder at ∼227 nm, which indicates an Mg²⁺-induced change in secondary structure. This Mg²⁺-specific effect is possibly crucial for the physiological role of PfN44 in the regulation of calcite/aragonite deposition.

Model peptides

Instead of looking at full-length proteins, numerous studies have investigated peptides that represent domains, which contain amino acid sequence patterns being typical for their capability to control nucleation.^{Reference Wustman, Morse and Evans55–Reference Ajikumar, Lakshminarayanan, Ong, Valiyaveettil and Kini59} Some of these peptides show nucleation properties in vitro. It is highly probable that their amino acid sequence and the secondary structure these peptides exhibit are the same as in domains that determine the functionality of the corresponding full-length proteins. The C- and N-terminal ends of the nacre-associated oyster shell protein n16 from Pinctada fucata contain sequence motifs that are generally associated with Ca²⁺ binding.^{Reference Samata, Hayashi, Kono, Hasegawa, Horita and Akera60} The 30 amino acid peptides n16-N and n16-C representing these two domains both modulate the morphology of calcium carbonate crystals in vitro.^{Reference Kim, DiMasi and Evans61} A strong negative band at 200 nm in the CD spectra of 4 µM solutions of n16-N and n16-C indicates unordered conformation (Figure 4c). However, at higher concentration (8 µM), a negative band appears at 215 nm, which can be attributed to formation of β-strand conformation. The positive band showing up at 233 nm for n16-N, however, is not associated with a β-sheet. Nevertheless, the significantly different appearance of CD spectra recorded for different peptide concentrations is a clear sign of intermolecular interaction. As aggregation effects have also been observed for BPPs from different organisms, the propensity for protein/protein-assembly may be mediated by amino acid sequences that are typical for Ca²⁺ binding.

Effects of temperature

According to its CD spectrum, the recombinant full-length enamel protein porcine amelogenin rP172 adopts a predominantly unordered structure in the presence of Ca²⁺ ions, indicated by a strong minimum at 200 nm and a weak positive shoulder at ∼220 nm (Figure 5a).^{Reference Lakshminarayanan, Yoon, Hegde, Fan, Du and Moradian-Oldak64} Increased temperature leads to a shift of the minimum and a decrease of the shoulder. As already discussed for phosvitin, this indicates decreasing content of P_II-helix. Interestingly, all CD curves in Figure 5a intersect at one particular point at ∼211 nm. The existence of such an isoelliptoic point (referring to constant ellipticity, a synonym for CD intensity) indicates a transition between two states. The linear decrease of the intensity at ∼220 nm together with an isoelliptoic point is typical for a temperature-dependent transition from a P_II-helix to a β-strand structure.^{Reference Lakshminarayanan, Yoon, Hegde, Fan, Du and Moradian-Oldak64–Reference Makowska, Rodziewicz-Motowidlo, Baginska, Vila, Liwo, Chmurzynski and Scheraga66}

Figure 5.

(a) Amelogenin (rP172) at different temperatures in the presence of 5 mM CaCl₂. Temperature increase is accompanied by a transition from unordered to β-sheet conformation. (b) rP172 in the presence of lipid vesicles or sodium dodecyl sulfate (SDS) micelles at pH 3.5. Negatively charged lipids (POPG, ACML) invoke aggregation. POPC has no effect, and SDS induces β-sheet. (c) Mixture of peptides pA and pB, representing domains of DMP1, with and without Ca²⁺. Addition of Ca²⁺ induces assembly and β-sheet. (a) Adapted with permission from Reference 64. (b) rP172 adapted with permission from Reference 68. SDS micelles adapted with permission from Reference 69. (c) Adapted with permission from Reference 74.

Binding to lipid vesicles

A hallmark of BPPs is the high abundance of acidic residues, suggesting putative effects from protein/cation interactions. However, during the mineralization process, they may also interact with negatively charged surfaces such as lipid membranes, which may have implications for protein structure. This has been described for amelogenin, where the presence of membranes of a particular lipid composition seems to have an impact protein assembly.^{Reference Chen, Bromley, Moradian-Oldak and De Yoreo67}

For studies on membrane effects, the protein of interest can be mixed with liposomes, which are vesicles composed of lipid bilayers frequently used to mimic a cell membrane in biophysical investigations. Amelogenin rP172 has been tested in the presence of liposomes that consisted either of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), or a mixture of different lipids chosen to mimic the composition of ameloblast membranes (ameloblast cell membrane-mimicking lipid vesicles, ACML).^{Reference Lokappa, Chandrababu, Dutta, Perovic, Evans and Moradian-Oldak68} While the presence of zwitterionic POPC liposomes does not seem to affect the CD spectrum of rP172, the spectra look significantly different in the presence of negatively charged POPG and ACML (Figure 5b). Based on the appearance of the CD spectrum and findings from NMR experiments, which suggest lipid-induced changes of the protein backbone, this was interpreted as a transition to α-helical conformation.^{Reference Lokappa, Chandrababu, Dutta, Perovic, Evans and Moradian-Oldak68} However, the CD spectra recorded in presence of POPG and ACML also resemble a dispersive curve shape spectrum of particles that contain a protein in an unordered conformation (Figure 1b). Furthermore, the distribution of hydrodynamic radii determined by dynamic light scattering analyses is slightly different for rP172-POPG and rP172-ACML in favor of larger-sized particles compared to rP172-POPC mixtures.^{Reference Lokappa, Chandrababu, Dutta, Perovic, Evans and Moradian-Oldak68} This can be interpreted as a different propensity for protein-protein or protein-lipid assembly for different lipid compositions. Interestingly, while there are significant lipid dependent changes to the CD spectrum at pH 3.5, there are only minor effects observed at pH 8.^{Reference Lokappa, Chandrababu, Dutta, Perovic, Evans and Moradian-Oldak68}

Binding to detergent micelles

Sodium dodecyl sulfate (SDS) is a detergent that forms negatively charged micelles. The addition of SDS above its critical micellar concentration to the amelogenin rP172 has led to significant changes in the CD spectrum involving a shift of the characteristic minimum from 200 nm to 208 nm and the formation of a negative shoulder around 220 nm (Figure 5b).^{Reference Chandrababu, Dutta, Lokappa, Ndao, Evans and Moradian-Oldak69} These spectral features have been associated with the formation of a β-sheet in other BPPs (see previous text). This agrees with reports that SDS, in spite of its use as a denaturing agent for gel electrophoresis, can sometimes induce local secondary structures in intrinsically disordered proteins.^{Reference Otzen70} Different from the effects observed for liposomes, the spectral changes observed for the addition of SDS have not been pH-dependent. As the pH-dependent conformational equilibrium in rP172 seems to be nullified by SDS, the interaction with highly charged detergent micelles is probably not viable as a model for the physiological situation.

Model peptides

It has been postulated that the acidic phosphoprotein DMP1 plays a role in calcium binding and mineralization.^{Reference George, Sabsay, Simonian and Veis71,Reference George, Silberstein and Veis72} Like other matrix proteins, it contains acidic domains that can possibly interact with calcium.^{Reference George, Sabsay, Simonian and Veis71} Two short peptides representing such domains (pA and pB) were synthesized and characterized.^{Reference He, Dahl, Veis and George73} While neither of these peptides alone has been capable of initiating the crystallization of apatite, a mixture of both induced formation of plate-shaped apatite crystals in vitro, which was concomitant with peptide oligomerization.^{Reference He, Dahl, Veis and George73} Similar to full-length BPPs, CD spectra of the pA/pB mixture exhibit the characteristic minimum at 200 nm typical of unordered conformation (Figure 5c). In the presence of Ca²⁺, a minimum around 220 nm indicates the emergence of β-sheet conformation. As β-sheet conformation is generally associated with a higher propensity for self-aggregation,^{Reference Cheng, Pham and Nowick8} the Ca²⁺-induced conformational change is possibly driving the self-assembly of pA and pB peptides. It has been hypothesized that self-assembly was a prerequisite for DMP-induced mineralization. This assumption is supported by the fact that CD spectra of the related DMP4 show very similar Ca²⁺-induced spectral changes as the DMP1-derived acidic domain peptides pA and pB.^{Reference Hao, Narayanan, Muni, Ramachandran and George74}