Marie-Louise Lemloh

Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization

Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO2 mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals.

Biomineralization in Ciliates

From a biomineralization point of view, the protist world is far less investigated than its metazoan counterpart. However, eukaryotic single-celled organisms offer a very unique access to discover biomineralization mechanisms in vivo. With respect to intracellular mechanisms involved in ion enrichment, mineral transport or vesicle formation ciliates represent a good model system. One important group of protists, the ciliates, is very common and numerous studies have been performed on their ecology, cell biology, morphology or genetics. Ciliates are also known for their formation of diverse mineralized intracellular and extracellular structures. However, only limited numbers of detailed studies on the kind of minerals, their properties or their formation mechanisms have been reported so far. This article reviews older and more recent literature on biomineralization in ciliates.

Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora, Prostomatea).

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.

Isolation of alveolar plates from Coleps hirtus.

In the ciliate Coleps hirtus, the alveoli contain rigid alveolar plates that are almost unstudied so far. Neither the exact composition nor the genesis and function are known. A necessary step to study the alveolar plates is to isolate these structures in an adequate amount. Therefore, culture conditions of C. hirtus were optimized to obtain an axenic and dense long-time culture. The protocol we developed to isolate C. hirtus alveolar plates is presented and clean alveolar plates were documented via scanning electron microscopy. The described procedure delivers alveolar plates of very good structure and integrity with preserved filigree details in sufficient amount. They can be analysed via a range of different material and biological characterisations. Since there are indications of a mineral phase within the alveolar plates, the presented results will allow to study C. hirtus alveolar plates also in the context of biomineralisation.

Aqueous ball milling of nacre constituents facilitates directional self-assembly of aragonite nanoparticles of the gastropod Haliotis glabra

A ball-milling approach was developed to investigate the constituents of isolated nacre tablets of the gastropod Haliotis glabra in aqueous suspension without additional chemical additives. The obtained particle mixtures were characterized using X-ray crystallography as well as scanning and transmission electron microscopy. Aragonite nanoparticles retained their crystal structure even after 14 h of ball milling. The long-term stability of the particle mixtures varied as a function of the ball-milling duration. An increased milling time led to rod-like stable assemblies of aragonite nanoparticles. Selected area electron diffraction investigations revealed that the longitudinal axes in about one-third of these nanoparticle rods were oriented along the crystallographic c-axis of aragonite, indicating oriented attachment of the aragonite nanoparticles. These in vitro observations support the idea that a two-stage process, separated into crystallization of nanoparticles and oriented assembly of nanocrystals, could also occur in vivo.

Sulphur-Containing Compounds as a Response in Sea Urchins Exposed to Alkylated Silicon Nanocrystals and SiO2-Coated Iron Oxide Nanoparticles

We report the effects of exposure to alkylated silicon nanocrystals (‘alkyl-SiNCs’ at concentration ~ 7.2 mg/L) and -Fe2O3 nanoparticles coated with ultra-thin silica (‘SiO2-coated IONPs’ at concentration ~ 150 mg/L) on sea urchins Paracentrotus lividus and Arbacia lixula, respectively, studied with X-ray fluorescence (XRF) and Fourier transform infrared (FTIR) spectroscpoies using excitation from a synchrotron light source. A remarkably low mortality and low incidence of skeletal deformation is observed for exposure to both types of nanoparticles studied, despite the high concentrations employed in this work. XRF mapping demonstrates that both types of nanoparticle are found to agglomerate in the body of the sea urchins. FTIR spectra indicates that alkyl-SiNCs remain intact after ingestion and corresponding XRF maps show increased an oxygen throughout the organisms, possibly related to oxidation products arising from reactive oxygen species generated in the presence of the nanoparticles. Exposure to SiO2-coated IONPs is found to produce sulphur-containing species, which may be the result of a biological response in order to reduce the toxicity of the nanomaterial.