Effect of Surface Hydrophobicity on the Function of the Immobilized Biomineralization Protein Mms6 (original) (raw)
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Adsorption of Biomineralization Protein Mms6 on Magnetite (Fe3O4) Nanoparticles
International Journal of Molecular Sciences
Biomineralization is an elaborate process that controls the deposition of inorganic materials in living organisms with the aid of associated proteins. Magnetotactic bacteria mineralize magnetite (Fe3O4) nanoparticles with finely tuned morphologies in their cells. Mms6, a magnetosome membrane specific (Mms) protein isolated from the surfaces of bacterial magnetite nanoparticles, plays an important role in regulating the magnetite crystal morphology. Although the binding ability of Mms6 to magnetite nanoparticles has been speculated, the interactions between Mms6 and magnetite crystals have not been elucidated thus far. Here, we show a direct adsorption ability of Mms6 on magnetite nanoparticles in vitro. An adsorption isotherm indicates that Mms6 has a high adsorption affinity (Kd = 9.52 µM) to magnetite nanoparticles. In addition, Mms6 also demonstrated adsorption on other inorganic nanoparticles such as titanium oxide, zinc oxide, and hydroxyapatite. Therefore, Mms6 can potentially...
Living organisms can produce inorganic materials with unique structure and properties. The biomineralization process is of great interest as it forms a source of inspiration for the development of methods for production of diverse inorganic materials under mild conditions. Nonetheless, regulation of biomineralization is still a challenging task. Magnetotactic bacteria produce chains of a prokaryotic organelle comprising a membrane-enveloped single-crystal magnetite with species-specific morphology. Here, we describe regulation of magnetite biomineralization through controlled expression of the mms7 gene, which plays key roles in the control of crystal growth and morphology of magnetite crystals in magnetotactic bacteria. Regulation of the expression level of Mms7 in bacterial cells enables switching of the crystal shape from dumbbell-like to spherical. The successful regulation of magnetite biomineralization opens the door to production of magnetite nanocrystals of desired size and morphology. By means of organic molecules, organisms produce finely tuned inorganic materials, even nanoscale structures 1–4. The relevant bioprocesses, collectively called biomineralization, are especially attractive to materials scientists because these processes also may facilitate the development of technologies for production of a wide variety of inorganic materials under mild conditions and in aqueous environments 5–11. In recent comprehensive molecular studies, proteins and the corresponding genes that are involved in biomineralization have been characterized in various biological systems 12–14. Genetically programmed expression of these proteins allows for highly regulated synthesis of materials in each organism 15. Thus, by regulating gene expression of key molecules in a biological system, researchers can create a process for production of inorganic materials with desired structure and morphological features. Magnetotactic bacteria synthesize a unique intracellular organelle, the magnetosome, composed of a mag-netite (Fe 3 O 4) or greigite (Fe 3 S 4) crystal enveloped in a lipid membrane 16. This biomineral has a size range of approximately 20–100 nm and a species-specific morphology, such as cuboctahedral, elongated prismatic, or bullet-shaped 17–19. Given that magnetite nanocrystal-producing bacteria are prokaryotes, development of genetic modification protocols is relatively easy. In fact, the methods for transformation 20 , specific gene deletion 21,22 , and induction of target gene expression 23,24 have already been developed for magnetotactic bacteria. The use of these approaches in proteomic and genetic studies 25–27 to elucidate the molecular mechanisms of magnetite biominer-alization helped to identify complex multistep pathways: localization of proteins 28,29 , invagination of the membrane 30 , vesicle formation 31 , actin filament formation 32 , assembly of vesicles onto the filament structure 33 , iron transport 34 , redox control 35 , crystal growth and morphological regulation 30,36–39. In particular, identification of several proteins involved in the morphological regulation of magnetite crystal suggested that crystal morphology
2011
Highly ordered mineralized structures created by living organisms are often hierarchical in structure with fundamental structural elements at nanometer scales. Proteins have been found responsible for forming many of these structures, but the mechanisms by which these biomineralization proteins function are generally poorly understood. To better understand its role in biomineralization, the magnetotactic bacterial protein, Mms6, which promotes the formation in vitro of superparamagnetic magnetite nanoparticles of uniform size and shape, was studied for its structure and function. Mms6 is shown to have two phases of iron binding: one high affinity and stoichiometric and the other low affinity, high capacity, and cooperative with respect to iron. The protein is amphipathic with a hydrophobic N-terminal domain and hydrophilic C-terminal domain. It self-assembles to form a micelle, with most particles consisting of 20−40 monomers, with the hydrophilic C-termini exposed on the outside. Studies of proteins with mutated C-terminal domains show that the C-terminal domain contributes to the stability of this multisubunit particle and binds iron by a mechanism that is sensitive to the arrangement of carboxyl/hydroxyl groups in this domain.
Chemistry (Weinheim an der Bergstrasse, Germany), 2016
Formation of magnetite nanocrystals by magnetotactic bacteria is controlled by specific proteins which regulate the particles' nucleation and growth. One such protein is Mms6. This small, amphiphilic protein can self-assemble and bind ferric ions to aid in magnetite formation. To understand the role of Mms6 during in vitro iron oxide precipitation we have performed in situ pH titrations. We find Mms6 has little effect during ferric salt precipitation, but exerts greatest influence during the incorporation of ferrous ions and conversion of this salt to mixed-valence iron minerals, suggesting Mms6 has a hitherto unrecorded ferrous iron interacting property which promotes the formation of magnetite in ferrous-rich solutions. We show ferrous binding to the DEEVE motif within the C-terminal region of Mms6 by NMR spectroscopy, and model these binding events using molecular simulations. We conclude that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ion...
Magnetotactic bacteria that produce magnetic nanocrystals of uniform size and well-defined morphologies have inspired the use of biomineralization protein Mms6 to promote formation of uniform magnetic nanocrystals in vitro. Small angle X-ray scattering (SAXS) studies in physiological solutions reveal that Mms6 forms compact globular threedimensional (3D) micelles (approximately 10 nm in diameter) that are, to a large extent, independent of concentration. In the presence of iron ions in the solutions, the general micellar morphology is preserved, however, with associations among micelles that are induced by iron ions. Compared with Mms6, the m2Mms6 mutant (with the sequence of hydroxyl/carboxyl containing residues in the Cterminal domain shuffled) exhibits subtle morphological changes in the presence of iron ions in solutions. The analysis of the SAXS data is consistent with a hierarchical core−corona micellar structure similar to that found in amphiphilic polymers. The addition of ferric and ferrous iron ions to the protein solution induces morphological changes in the micellar structure by transforming the 3D micelles into objects of reduced dimensionality of 2, with fractal-like characteristics (including Gaussian-chain-like) or, alternatively, plateletlike structures.
Crystal Growth & Design, 2017
MamC from Magnetococcus marinus MC-1 has been shown to control the size of magnetite crystals in in vitro experiments, thereby demonstrating its potential as a candidate protein for the production of magnetite nanoparticles possibly useful in medical and other applications. However, the importance of the structure and aggregation state of the protein on the resulting biomimetic nanoparticles has not yet been assessed. One method normally used to prevent the aggregation of integral membrane proteins is the introduction of detergents during protein purification. In this study, results from protein aggregation following the addition of Triton-X100, DDM, and LDAO are presented. Magnetite particles formed in the presence of MamC purified using these three detergents were compared. Our results show that detergents alter the structure of the folded recombinant protein, thus preventing the ability of MamC to control the size of magnetite crystals formed chemically in vitro. Furthermore, we show that the introduction of detergents only at the dialysis process during the protein purification prevents its aggregation and allows for correct, functional folding of MamC. These results also indicate that the population of the active protein particles present at a certain oligomeric state needs to be considered, rather than only the oligomeric state, in order to interpret the ability of magnetosome recombinant proteins to control the size and/or morphology of magnetite crystals formed chemically in vitro.
Self-assembled MmsF proteinosomes control magnetite nanoparticle formation in vitro
Proceedings of the National Academy of Sciences of the United States of America, 2014
Magnetotactic bacteria synthesize highly uniform intracellular magnetite nanoparticles through the action of several key biomineralization proteins. These proteins are present in a unique lipid-bound organelle (the magnetosome) that functions as a nanosized reactor in which the particle is formed. A master regulator protein of nanoparticle formation, magnetosome membrane specific F (MmsF), was recently discovered. This predicted integral membrane protein is essential for controlling the monodispersity of the nanoparticles in Magnetospirillum magneticum strain AMB-1. Two MmsF homologs sharing over 60% sequence identity, but showing no apparent impact on particle formation, were also identified in the same organism. We have cloned, expressed, and used these three purified proteins as additives in synthetic magnetite precipitation reactions. Remarkably, these predominantly α-helical membrane spanning proteins are unusually highly stable and water-soluble because they self-assemble into...
Biomaterials, 2007
Mms6 is a small acidic protein that is tightly associated with bacterial magnetite in Magnetospirillum magneticum AMB-1. This protein has previously shown iron binding activity, allowing it to generate uniform magnetic crystals by co-precipitation of ferrous and ferric ions. Here, magnetite crystals were formed by the partial oxidation of ferrous hydroxide in the presence and absence of Mms6. The crystals synthesised were systematically characterised according to their sizes and morphologies using high-resolution transmission electron microscopy. Mms6-mediated synthesis of magnetite by this methods produced crystals of a uniform size and narrow size distribution with a cubo-octahedral morphology, similar to bacterial magnetite observed in M. magneticum AMB-1. The crystals formed in the absence of Mms6 were octahedral, larger with an increased size distribution. Protein quantification analysis of Mms6 in the synthesised particles indicated tight association of this protein onto the crystal. Furthermore, high affinities to iron ions and a highly charged electrostatic quality suggest that the protein acts as a template for the nucleus formation and/or acts as a growth regulator by recognising crystal faces. The method introduced in this study presents an alternative route for controlling the size and shape of magnetite crystals without the use of organic solvent and high temperatures.
Journal of Materials Chemistry, 2011
Magnetic nanoparticles (MNPs) are in high demand within biomedical and nanotechnological industries. Size, shape, material and crystal quality directly affect the particle's properties, namely their magnetic characteristics, and must be tuned and controlled to meet the specification of the application. A key challenge is to refine synthetic methods to tailor the MNP properties with precision, but using cheap, high-yield, industrially robust and environmentally friendly methods. In this study we compare simple high-yield precipitation methods of producing cobalt-doped magnetite MNPs. We explore the variation of magnetic coercivity and saturation with increasing Co-doping from 0-15% in magnetite MNPs, which increases coercivity from 5-62 mT, but decreases saturation from 91-28 emu g À1. An optimum of 6% was further investigated as this produced the greatest increase in coercivity to 34 mT with a relatively small reduction in saturation magnetisation to 79 emu g À1. The methods compared are refined with the addition of the recombinant biomineralisation protein Mms6 from a magnetic bacterium, as this has been shown to help control magnetite MNP morphology and grainsize distribution in vitro. Similar control is seen here over our Co-doped magnetite synthesis. Mms6 increases the size and decreases the size distribution of room temperature co-precipitated particles from 11.7 nm to 31.7 nm. The affinity tagged protein his 6 Mms6 also controls the size (23.2 nm) but less effectively than Mms6. Therefore the Mms6 mediated Co-doped MNP particles are found to be single domain and thus give very clear, square magnetic hysteresis with a coercivity of 48 mT at 10 K. Hysteresis of the smaller particles (Co-doped MNP with no protein and with his-tagged protein) clearly shows both superparamagnetic and single-domain magnetic behaviours. Powder X-ray diffraction shows that both the addition of Mms6 and cobalt increases the crystal quality of the MNP. Thus Mms6 protein mediated room temperature co-precipitation offers an environmentally friendly, industrially robust route towards tailored, uniform, single-domain, high-quality Co-doped magnetite MNPs.
Frontiers in Microbiology
Biomineralization is a process that takes place in all domains of life and which usually helps organisms to harden soft tissues by creating inorganic structures that facilitate their biological functions. It was shown that biominerals are under tight biological control via proteins that are involved in nucleation initiation and/or which act as structural skeletons. Magnetotactic bacteria (MTB) use iron biomineralization to create nano-magnetic particles in a specialized organelle, the magnetosome, to align to the geomagnetic field. A specific set of magnetite-associated proteins (MAPs) is involved in regulating magnetite nucleation, size, and shape. These MAPs are all predicted to contain specific 17-22 residue-long sequences involved in magnetite formation. To understand the mechanism of magnetite formation, we focused on three different MAPs, MamC, Mms6 and Mms7, and studied the predicted iron-binding sequences. Using nuclear magnetic resonance (NMR), we differentiated the recognition mode of each MAP based on ion specificity, affinity, and binding residues. The significance of critical residues in each peptide was evaluated by mutation followed by an iron coprecipitation assay. Among the peptides, MamC showed weak ion binding but created the most significant effect in enhancing magnetite particle size, indicating the potency in controlling magnetite particle shape and size. Alternatively, Mms6 and Mms7 had strong binding affinities but less effect in modulating magnetite particle size, representing their major role potentially in initiating nucleation by increasing local metal concentration. Overall, our results explain how different MAPs affect magnetite synthesis, interact with Fe 2+ ions and which residues are important for the MAPs functions.