Protein and Peptide Letters - Volume 25, Issue 1, 2018

Background: Various types of proteins play important roles in the biomineralization of hydroxyapatite (HAp, Ca10(PO4)6(OH)2). The resulting organic-HAp nanohybrids have highlyorganized hierarchical structures that show unique morphological, structural, and mechanical properties. By mimicking the biomineralization process, organic-HAp hybrid materials have been created by utilizing proteins and peptides. Objectives: In this review, firstly the roles of proteins in HAp mineralization in vivo are briefly explained. Recent progresses in the creation of organic-HAp hybrids through the utilization of proteins and peptides are then described. Results: Roles of collagen and amelogenin on the formation of bones and teeth were explained. Then, recent advances, including those by the authors, in the creation of organic-HAp hybrids through the utilization of these proteins, their derivatives, and synthetic peptides, including engineering- isolated ones, were reviewed. Conclusion: Organic-HAp hybrid materials have been intensively created by utilizing proteins and peptides. Among them, engineering-isolated or rationally designed peptides and their derivatives represent future promising building components for organic-HAp hybrids with precise hierarchical structures. Not only the excellent functions of the resultant hybrids materials, but also the creation of materials by biomimetic synthetic processes at a low cost and environmental burden are important for sustainable industrial development.

Background: A core sequence (the 9 C-terminal residues) of calcification-associated peptide (CAP- 1) isolated from the exoskeleton of the red swamp crayfish was previously shown to control calcium carbonate precipitation with chitin. In addition, a modified core sequence in which the phosphorylated serine at the N terminus is replaced with serine exhibits was also previously shown to alter precipitation characteristics with chitin. Objectives: We focused on calcium carbonate precipitation and attempted to elucidate aspects of the mechanism underlying mineralization. We attempted to evaluate in detail the effects of modifying the N-terminus in the core sequence on calcium carbonate mineralization without chitin. Methods: The peptide modifications included phosphorylation, dephosphorylation, and a free or acetylated Nterminus. The peptides were synthesized manually on Wang resin using the DIPCI-DMAP method for the first residue, and Fmoc solid phase peptide synthesis with HBTU-HOBt for the subsequent residues. Prior to calcium carbonate precipitation, calcium carbonate was suspended in MilliQ water. Carbon dioxide gas was bubbled into the stirred suspension, then the remaining solid CaCO3 was removed by filtration. The concentration of calcium ions in the solution was determined by standard titration with ethylenediaminetetraacetate. Calcium carbonate precipitation was conducted in a micro tube for 3 h at 37°C. We used the micro-scale techniques AFM (atomic force microscopy) and TEM (transmission electron microscopy), and the macro-scale techniques chelate titration, HPLC, gel filtration, CD (circular dichroism) and DLS (dynamic light scattering). Results: We determined the morphologies of the calcium carbonate deposits using AFM and TEM. The pS peptide provided the best control of the shape and size of the calcium carbonate round particles. The acetylated peptides (Ac-S and Ac-pS) provided bigger particles with various shapes. S peptide provided a mixture of bigger particles and amorphous particles. We verified these findings using DLS. All the peptide samples produced nanostructures of the expected size in agreement with the AFM and TEM results. We estimated the abilities of these peptides to precipitate calcium carbonate by determining the residual calcium hydrogen carbonate concentration by standard titration with ethylenediaminetetraacetate after calcium carbonate precipitation. The Ac-pS peptide showed the lowest residual calcium hydrogen carbonate concentration whereas the S peptide showed the highest, suggesting that the precipitating activities of these peptides towards calcium carbonate correlated with peptide net charge. Then the gel filtration results showed a large oligomer peak and a small oligomer/monomer peak for all peptide samples in agreement with the AFM, TEM and DLS results. CD measurements showed that all the peptides formed random-coil-like structures. Thus, we used both macro- and micro-observation techniques such as chelate titration, DLS, AFM and TEM to show that the calcium carbonate precipitating activities of four derivatives of the core sequence of CAP-1 may correlate with the peptide net charge. Conclusion: These peptides mainly act as a catalyst rather than as a binder or component of the calcium carbonate deposits (as a template). On the other hand, the morphologies of the calcium carbonate deposits appeared to be dependent on the ability of the peptide to assemble and act as a template. Consequently, elucidating the relationship between peptide sequence and the ability of the peptide to assemble would be indispensable for controlling precipitate morphologies in the near future. This knowledge would provide important clues for elucidating the relationship between peptide sequence and mineralization ability, including deposit morphology and precipitating activity, for use in nanobiochemistry and materials chemistry research.

Background: Gold nanocrystals have unique physicochemical and biocompatible properties, and hold promise for use as catalysts and in the fields of electronics, photonic and/or plasmonic devices, sensing and/or imaging systems, targeted drug delivery, and photothermal therapies. A variety of organic templates have been used to control the size, shape, and structure of gold nanocrystals, and to modify their surfaces. For the control of the shape of gold nanocrystals, we previously designed and synthesized a β-sheet-forming nonapeptide (RU006: Ac- AIAKAXKIA-NH2, X = L-2-naphthylalanine, Nal). A mixture of RU006 and HAuCl4 in water produced ultrathin gold nanoribbons with 50-100 nm wide, several nanometers high, and microns long. Objectives: The main objective of this study is the control of the nanoribbon crystal growth by designing and synthesizing RU006 analogs containing an N-methyl-L-alanine residue. Methods: We report (i) the design and synthesis of four RU006 analogs in which an L-alanine (Ala) at four positions in the RU006 sequence (N-methylated RU006 analogs) is replaced with an N-methyl alanine, (ii) conformational and morphological analyses of the self-assembled Nmethylated RU006 analogs, (iii) gold nanocrystal synthesis by the peptide templating method with N-methylated RU006 analogs, and (iv) the roles of peptide self-assembly in anisotropic gold crystal growth. Results: RU006 with an N-methyl moiety at the center position resulted in flattened/platelet gold nanocrystals. It was also found that decreasing the mole fraction of RU006 in mixtures with Nmethylated RU006 analogs afforded significantly different absorption spectra compared to that obtained using RU006 alone under gold nanocrystal synthesis conditions. Conclusion: We found that morphology of gdd nanocrystals is significantly affected by electron transfer from the naphthalene rings to HAuCl4, accompanied by cross-linking reactions between spatially adjacent naphthalene rings within the hydrophobic cavity of a template assembly.

Background: Mercury (II) ion, Hg2+ is among the most common pollutants with the ability to affect the environment. The implications of their elevation in the environment are mainly due to the industrialization and urbanization process. Current methods of Hg2+ detection primarily depend on sophisticated and expensive instruments. Hence, an alternative and practical way of detecting Hg2+ ions is needed to go beyond these limitations. Here, we report a detection method that was developed using an inhibitive enzymatic reaction that can be monitored through a smartphone. Horseradish peroxidase (HRP) converted 4-aminoantipyrene (4-AAP) into a red colored product which visible with naked eye. A colorless product, on the other hand, was produced indicating the presence of Hg2+ that inhibit the reaction. Objectives: The aim of this study is to develop a colorimetric sensor to detect Hg2+ in water sources using HRP inhibitive assay. The system can be incorporated with a mobile app to make it practical for a prompt in-situ analysis. Methods: HRP enzyme was pre-incubated with different concentration of Hg2+ at 37°C for 1 hour prior to the addition of chromogen. The mix of PBS buffer, 4-AAP and phenol which act as a chromogen was then added to the HRP enzyme and was incubated for 20 minutes. Alcohol was added to stop the enzymatic reaction, and the change of colour were observed and analyse using UV-Vis spectrophotometer at 520 nm wavelength. The results were then analysed using GraphPad PRISM 4 for a non-linear regression analysis, and using Mathematica (Wolfram) 10.0 software for a hierarchical cluster analysis. The samples from spectroscopy measurement were directly used for dynamic light scattering (DLS) evaluation to evaluate the changes in HRP size due to Hg2+ malfunctionation. Finally, molecular dynamic simulations comparing normal and malfunctioned HRP were carried out to investigate structural changes of the HRP using YASARA software. Results: Naked eye detection and data from UV-Vis spectroscopy showed good selectivity of Hg2+ over other metal ions as a distinctive color of Hg2+ is observed at 0.5 ppm with the IC50 of 0.290 ppm. The mechanism of Hg2+ inhibition towards HRP was further validated using a dynamic light scattering (DLS) and molecular dynamics (MD) simulation to ensure that there is a conformational change in HRP size due to the presence of Hg2+ ions. The naked eye detection can be quantitatively determined using a smartphone app namely ColorAssist, suggesting that the detection signal does not require expensive instruments to be quantified. Conclusion: A naked-eye colorimetric sensor for mercury ions detection was developed. The colour change due to the presence of Hg2+ can be easily distinguished using an app via a smartphone. Thus, without resorting to any expensive instruments that are mostly laboratory bound, Hg2+ can be easily detected at IC50 value of 0.29 ppm. This is a promising alternative and practical method to detect Hg2+ in the environment.