Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment assemblies and the evolution and the design of vertebrate mineralized tissue microstructures. Our findings reveal that in the greater context of chordate evolution, the biological control of apatite growth by polyproline-based matrix assemblies provides a molecular basis for the evolution of the vertebrate body plan.</p

Highly acidic pH facilitates enamel protein self-assembly, apatite crystal growth and enamel protein interactions in the early enamel matrix

Tooth enamel develops within a pH sensitive amelogenin-rich protein matrix. The purpose of the present study is to shed light on the intimate relationship between enamel matrix pH, enamel protein self-assembly, and enamel crystal growth during early amelogenesis. Universal indicator dye staining revealed highly acidic pH values (pH 3–4) at the exocytosis site of secretory ameloblasts. When increasing the pH of an amelogenin solution from pH 5 to pH 7, there was a gradual increase in subunit compartment size from 2 nm diameter subunits at pH 5 to a stretched configuration at pH6 and to 20 nm subunits at pH 7. HSQC NMR spectra revealed that the formation of the insoluble amelogenin self-assembly structure at pH6 was critically mediated by at least seven of the 11 histidine residues of the amelogenin coil domain (AA 46–117). Comparing calcium crystal growth on polystyrene plates, crystal length was more than 20-fold elevated at pH 4 when compared to crystals grown at pH 6 or pH 7. To illustrate the effect of pH on enamel protein self-assembly at the site of initial enamel formation, molar teeth were immersed in phosphate buffer at pH4 and pH7, resulting in the formation of intricate berry tree-like assemblies surrounding initial enamel crystal assemblies at pH4 that were not evident at pH7 nor in citrate buffer. Amelogenin and ameloblastin enamel proteins interacted at the secretory ameloblast pole and in the initial enamel layer, and co-immunoprecipitation studies revealed that this amelogenin/ameloblastin interaction preferentially takes place at pH 4—pH 4.5. Together, these studies highlight the highly acidic pH of the very early enamel matrix as an essential contributing factor for enamel protein structure and self-assembly, apatite crystal growth, and enamel protein interactions

Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (∼P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule

Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

How does proline-repeat motif length in the proteins of teeth and bones relate to the evolution of vertebrates? Counterintuitively, longer repeat stretches are associated with smaller aggregated subunits within a supramolecular matrix, resulting in enhanced crystal length in mammalian versus amphibian tooth enamel

Genetic analysis of Schizosaccharomyces pombe 7SL RNA

Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one molecule of 7SL RNA, is required for translocation of secretory proteins into mammalian microsomal vesicles in vitro. In order to investigate the function of the RNA component of SRP, mutations were introduced into the S. pombe 7SL RNA gene by both site-directed and random mutagenesis.The site-directed mutagenesis target was a 6-nucleotide segment within a 35-nucleotide structure which has been preserved in RNAs from many divergent organisms. This domain of 7SL RNAs interacts with two SRP proteins, the 19-kDa polypeptide and the 68/72-kDa heterodimer. The data demonstrated that, despite its conservation over vast evolutionary distances, many changes in this region can be tolerated under normal growth conditions. An exception is the lethality of several mutations at positions 159 and 160. Nucleotide 160 is the most highly conserved base in a single-stranded sequence which conforms to a consensus for the most common tetranucleotide loop in ribosomal RNAs. Mutations which are likely to affect the stability and/or conformation of the RNA give rise to a conditional phenotype: when osmolarity of the medium is raised, the RNAs become partially or completely defective in function at high temperature. The defects associated with mutations at position 159 can be partially rescued by mutating position 164 to restore base-pairing.In order to obtain high efficiency random mutagenesis localized to the 7SL coding sequence, a new method for forced nucleotide misincorporation in vitro was developed. Greater than 50% mutants can be generated over a large (200 nucleotide) DNA segment. The results from random mutagenesis show that S. pombe 7SL RNA is generally insensitive to point mutations. Fifty seven mutant alleles, many with multiple substitutions, have been transformed into fission yeast. Only five of these, four of which are located in the 35-nucleotide conserved segment, generated lethal phenotypes. A mutant 7SL RNA carrying 6 point mutations shows a temperature sensitive-phenotype. Because of its slow response after shift to the nonpermisive temperature, the defect is likely to be in synthesis or assembly of SRP, rather than in its function.U of I OnlyETDs are only available to UIUC Users without author permissio

Author Correction: Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

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