Il Won Kim

Regulation of in vitro calcium phosphate mineralization by combinatorially selected hydroxyapatite-binding peptides.

We report selection and characterization of hydroxyapatite-binding heptapeptides from a peptide-phage library and demonstrate the effects of two peptides, with different binding affinities and structural properties, on the mineralization of calcium phosphate mineral. In vitro mineralization studies carried out using one strong- and one weak-binding peptide, HABP1 and HABP2, respectively, revealed that the former exhibited a drastic outcome on mineralization kinetics and particle morphology. Strong-binding peptide yielded significantly larger crystals, as observed by electron microscopy, in comparison to those formed in the presence of a weak-binding peptide or in the negative control. Molecular structural studies carried out by circular dichroism revealed that HABP1 and HABP2 differed in their secondary structure and conformational stability. The results indicate that sequence, structure, and molecular stability strongly influence the mineralization activity of these peptides. The implication of the research is that the combinatorially selected short-sequence peptides may be used in the restoration or regeneration of hard tissues through their control over of the formation of calcium phosphate biominerals.

Probing the molecular mechanisms of quartz-binding peptides.

Understanding the mechanisms of biomineralization and the realization of biology-inspired inorganic materials formation largely depends on our ability to manipulate peptide/solid interfacial interactions. Material interfaces and biointerfaces are critical sites for bioinorganic synthesis, surface diffusion, and molecular recognition. Recently adapted biocombinatorial techniques permit the isolation of peptides recognizing inorganic solids that are used as molecular building blocks, for example, as synthesizers, linkers, and assemblers. Despite their ubiquitous utility in nanotechnology, biotechnology, and medicine, the fundamental mechanisms of molecular recognition of engineered peptides binding to inorganic surfaces remain largely unknown. To explore propensity rules connecting sequence, structure, and function that play key roles in peptide/solid interactions, we combine two different approaches: a statistical analysis that searches for highly enriched motifs among de novo designed peptides, and, atomistic simulations of three experimentally validated peptides. The two strong and one weak quartz-binding peptides were chosen for the simulations at the quartz (100) surface under aqueous conditions. Solution-based peptide structures were analyzed by circular dichroism measurements. Small and hydrophobic residues, such as Pro, play a key role at the interface by making close contact with the solid and hindering formation of intrapeptide hydrogen bonds. The high binding affinity of a peptide may be driven by a combination of favorable enthalpic and entropic effects, that is, a strong binder may possess a large number of possible binding configurations, many of which having relatively high binding energies. The results signify the role of the local molecular environment among the critical residues that participate in solid binding. The work herein describes molecular conformations inherent in material-specific peptides and provides fundamental insight into the atomistic understanding of peptide/solid interfaces.

Probing the Organic-Mineral Interface at the Molecular Level in Model Biominerals

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.

Proteome of Human Calcium Kidney Stones

OBJECTIVES Idiopathic calcium oxalate (CaOx) stones are believed to develop attached to papillary subepithelial deposits called Randalls plaques. Calcium phosphate (CaP) stones, conversely, are thought to arise within the inner medullary collecting ducts, enlarging and damaging surround tubular structures as they expand. If this is true, we theorize that differences will be seen within the organic portion (matrix) of CaOx stones compared with CaP stones using a mass spectroscopy (MS) approach. METHODS From a cohort of 47 powdered stones, 25 calculi (13 CaOx, 12 CaP) were confirmed to contain a dominant mineral content of >80% by powder x-ray diffraction. Matrix proteins were then extracted, purified, and digested. Peptide tandem MS data were acquired, and spectra were searched against a large human protein database to identify protein matches. RESULTS No significant differences were seen between pattern profiles of CaOx and CaP stones. However, variations in protein expression patterns were seen within individual CaOx (monohydrate and dihydrate) and CaP (apatite and brushite) mineral subtypes, suggesting a relationship between crystal-surface binding properties and matrix composition. Both groups contain a large number of inflammatory proteins and a catalog of common proteins is included. CONCLUSIONS Calcium kidney stone matrix contains hundreds of proteins and is predominated by proteins associated with inflammatory response. Many of the same proteins were identified in both CaOx and CaP stones, suggesting inflammation as a unifying origin or a common secondary role in calcium stone pathogenesis.

Integrative design of a poly(ethylene glycol)-poly(propylene glycol)-alginate hydrogel to control three dimensional biomineralization

A mineralized polymeric matrix has been extensively studied to understand biomineralization processes and to further regulate phenotypic functions of various cells involved in osteogenesis and physiological homeostasis. It has been often proposed that several matrix variables including charge density, hydrophobicity, and pore size play vital roles in modulating composition and morphology of minerals formed within a three dimensional (3D) matrix. However, the aspects have not yet been systematically examined because a tool enabling the independent control of the matrix variables is lacking. This study presents an advanced integrative strategy to control morphology and composition of biominerals with matrix properties, by using a hydrogel formulated to independently control charge density, hydrophobicity, and porosity. The hydrogel consists of poly(ethylene glycol) monomethacrylate (PEGmM), poly(propylene glycol) monomethacrylate (PPGmM), and methacrylic alginate (MA), so the charge density and hydrophobicity of the hydrogel can be separately controlled with mass fractions of MA and PPGmM. Also, hydrogels which present only nano-sized pores, termed nanoporous hydrogels, are lyophilized and rehydrated to prepare the hydrogels containing micro-sized pores, termed microporous hydrogels. We find that increasing the mass fractions of MA and PPGmM of the microporous hydrogel promotes the growth of apatite layers because of the increases in the charge density, hydrophobicity and pore size. In contrast, increasing mass fractions of MA and PPGmM of the nanoporous hydrogel enhances the formation of calcium carbonate minerals. The dependency of the mineralization on hydrogel variables is related to the change in supersaturation of mineral ions. Overall, the results of this study will be highly useful to better understand the interplay of matrix variables in biomineralization and to design a wide array of mineralized matrix potentially used in cell therapies and tissue engineering.

Thin Films of Highly Planar Semiconductor Polymers Exhibiting Band-like Transport at Room Temperature.

We report the observation of band-like transport from printed polymer thin films at room temperature. This was achieved from donor-acceptor type thiophene-thiazole copolymer that was carefully designed to enhance the planarity of the backbone and the resulting transfer integral between the macromolecules. Due to the strong molecular interaction, the printed polymer film exhibited extremely low trap density comparable to that of molecular single crystals. Moreover, the energy barrier height for charge transport could be readily reduced with the aid of electric field, which led formation of extended electron states for band-like charge transport at room temperature.

Proteomic analysis of a matrix stone: a case report

Matrix stones are radiolucent bodies that present as soft muco-proteinaceous material within the renal collecting system. Following wide-angle X-ray diffraction (XRD) and scanning electron microscopy (SEM), we homogenized a surgically removed matrix stone, extracted and purified protein, and analyzed samples using tandem mass spectrometry for proteomic composition. Resulting spectra were searched using ProteinPilot 2.0, and identified proteins were reported with >95% confidence. Primary XRD mineral analysis was a biological apatite, and SEM revealed fibrous, net-like laminations containing bacterial, cellular, and crystalline material. Of the 33 unique proteins identified, 90% have not been previously reported within matrix stones and over 70% may be considered inflammatory or defensive in nature. Characterization of other matrix stone proteomes, in particular from non-infectious populations, may yield insights into the pathogenesis of this rare stone as well as the mineralogical process that occurs within crystalline calculi.

Constitutional isomers of a C3-symmetric molecule showing different piezochromic behaviours: on–off switching and colour tuning

The piezochromic behaviours of C3-symmetric molecules with p-bromophenyl side groups connected to a phenyl ring core through cyano-vinylene bridges were studied. Upon grinding, α-BPAN-Br having cyano groups at the α-position to the phenyl ring core exhibited substantial quenching of a bluish green emission (on–off switching) and its constitutional isomer, β-BPAN-Br, showed an emission colour change from bluish green to deep blue (colour tuning). When the molecules were exposed to an organic vapour, the initial emission of each molecule was restored. The powder X-ray diffraction and DSC studies revealed that the as-synthesized and vapour-annealed samples had the same crystalline structures, while the ground samples had amorphous structures. Their structural analogues, α-BPAN-H and β-BPAN-H which had no bromo groups, did not show any piezochromic or vapochromic behaviour. Poly(β-BPAN) consisting of covalently linked β-BPAN units were prepared by Ullmann reaction of β-BPAN-Br. The polymer showed similar luminescence to that of crystalline β-BPAN-Br, but its initial emission was not changed by grinding.

Effects of Humidity and Surfaces on the Melt Crystallization of Ibuprofen

Melt crystallization of ibuprofen was studied to understand the effects of humidity and surfaces. The molecular self-assembly during the amorphous-to-crystal transformation was examined in terms of the nucleation and growth of the crystals. The crystallization was on Al, Au, and self-assembled monolayers with –CH3, –OH, and –COOH functional groups. Effects of the humidity were studied at room temperature (18–20 °C) with relative humidity 33%, 75%, and 100%. Effects of the surfaces were observed at −20 °C (relative humidity 36%) to enable close monitoring with slower crystal growth. The nucleation time of ibuprofen was faster at high humidity conditions probably due to the local formation of the unfavorable ibuprofen melt/water interface. The crystal morphologies of ibuprofen were governed by the nature of the surfaces, and they could be associated with the growth kinetics by the Avrami equation. The current study demonstrated the effective control of the melt crystallization of ibuprofen through the melt/atmosphere and melt/surface interfaces.

Cooperative Modulation of Mineral Growth by Prismatic-Associated Asprich Sequences and Mg(II)

Cooperative effects of magnesium ions and acidic polypeptides originating from a family of proteins known as Asprich (mollusk Atrina rigida) were studied. In our previous studies, these two acidic polypeptides were found to be effective in controlling the morphology of the calcium carbonate mineral, the main inorganic constituent of prismatic layer of the mollusk shell. Since these Asprich sequences are believed to contain a putative magnesium binding domain, the morphology-controlling effects were further investigated with the addition of magnesium ions. The mineral morphology was dramatically changed by the combined influence of each polypeptides and the magnesium ions, substantiating the recognized importance of magnesium in the formation of calcium carbonate-based biominerals.