Biomineralization Proteins Research Articles

Glycosylation: A “Last Word” in the Protein-Mediated Biomineralization Process

Post-translational modifications are one way that biomineral-associated cells control the function and fate of proteins. Of the ten different types of post-translational modifications, one of the most interesting and complex is glycosylation, or the covalent attachment of carbohydrates to amino acid sidechains Asn, Ser, and Thr of proteins. In this review the author surveys some of the known biomineral-associated glycoproteins and summarizes recent in vitro recombinant protein experiments which test the impact of glycosylation on biomineralization protein functions, such as nucleation, crystal growth, and matrix assembly. These in vitro studies show that glycosylation does not alter the inherent function of the polypeptide chain; rather, it either accentuates or attenuates functionality. In essence, glycosylation gives the cell the “last word” as to what degree a biomineralization protein will participate in the biomineralization process.

Bone morphogenetic proteins in biomineralization of two endodontic restorative cements.

To assess the effect of biodentine (BD) and MTA-angelus (MTA) on biocompatibility, BMP2, BMP4, and osteocalcin (OC) expression. Subcutaneously implanted tubes of four groups (MTA, BD, Control, and Sham) were kept over 15, 30, and 60 days; histological analyses were performed using H&E and Von Kossa; ELISA quantified IL-1β and IL-8 expression; and qRT-PCR verified gene expression of BMPs and OC. Sham showed slight changes in profile/intensity of inflammatory infiltrate in all periods. Control had an inflammatory score significantly higher than Sham at 15 days (p < .05). BD revealed a similar inflammatory response to Sham, without significant changes over periods. MTA group exhibited an increase in chronic inflammatory profile at 30 days, with significant reduction at 60 days, when compared to Sham (p < .05). At 30/60 days, experimental groups presented birefringent areas. At 30/60 days, BD and MTA significantly increase IL-1β compared to Control, whereas an increase in IL-8 was observed only in BD. At 30/60 days, BD produces an expression of BMP2 whereas MTA influenced BMP4 and OC. Materials tested are biocompatible and they have osteoinductive activity; the materials influenced the expression of the tested mediators differently, suggesting different affinities with the substrate and the dental substrates.

Oligo(l-glutamic acids) in Calcium Phosphate Precipitation: Chain Length Effect.

The understanding of calcium phosphate precipitation is of major interest in different fields of science, including medicine, biomaterials, and physical chemistry. The presence of additive biomacromolecules has been known to influence various stages of the precipitation process from nucleation to crystal growth. In the current work, well-defined sequences of short, negatively charged peptides, oligo(l-glutamic acids), were utilized as a model, inspired by contiguous sequences of acidic amino acids in natural biomineralization proteins. The precipitate morphology and phases, the element time profile in solution and in the precipitates, as well as the kinetics during the precipitation process were analyzed to explain the effect of these short peptides on calcium phosphate precipitation. The results show that peptides can delay the phase transformation of an amorphous precursor phase to hydroxyapatite and that there is an optimal chain length for this effect at a given concentration of peptide. This study is the first part of a two-part series and is followed by a subsequent work to reveal the mechanism by which these short peptides influence the calcium phosphate precipitation.

A novel matrix protein PfX regulates shell ultrastructure by binding to specific calcium carbonate crystal faces

Here, we have identified a novel matrix protein, named PfX, from the pearl oyster Pinctada fucada, and investigated the effects of recombinant PfX protein on calcium carbonate crystallization. The expression of PfX was spatially concentrated in the mantle tissue and gill, the former of which is responsible for the formation of shell structures. The shell notching assay showed a PfX expression response during injured shell repair and regeneration, suggesting the potential involvement of this matrix protein in shell biomineralization. Further, an in vitro crystallization assay showed that PfX could alter the CaCO3 morphologies of both calcite and aragonite polymorphs. Correspondingly, a binding assay indicated that PfX has strong binding affinity for CaCO3 crystals, especially aragonite. Further, the protein's calcite binding capacity increased obviously when particular crystal faces were induced. In addition, PfX conjugated with fluorescent dye cyanine-5 (cy5) was preferentially distributed on rough crystal faces instead of the smooth and common (1 0 4) faces of calcite during the crystallization. These results suggest that matrix protein PfX might regulate CaCO3 morphology via selective binding and inhibit the growth of certain crystal faces, providing new clues for understanding biomineralization mechanisms in mollusk.

Molecular characterization of a whirlin-like protein with biomineralization-related functions from the shell of Mytilus coruscus.

Mollusc shells are produced from calcified skeletons and have excellent mechanical properties. Shell matrix proteins (SMPs) have important functions in shell formation. A 16.6 kDa whirlin-like protein (WLP) with a PDZ domain was identified in the shell of Mytilus coruscus as a novel SMP. In this study, the expression, function, and location of WLP were analysed. The WLP gene was highly expressed and specifically located in the adductor muscle and mantle. The expression of recombinant WLP (rWLP) was associated with morphological change, polymorphic change, binding ability, and crystallization rate inhibition of the calcium carbonate crystals in vitro. In addition, an anti-rWLP antibody was prepared, and the results from immunohistochemistry and immunofluorescence analyses revealed the specific location of the WLP in the mantle, adductor muscle, and myostracum layer of the shell, suggesting multiple functions for WLP in biomineralization, muscle-shell attachment, and muscle attraction. Furthermore, results from a pull-down analysis revealed 10 protein partners of WLP in the shell matrices and a possible network of interacting WLPs in the shell. In addition, in this study, one of the WLP partners, actin, was confirmed to have the ability to bind WLP. These results expand the understanding of the functions of PDZ-domain-containing proteins in biomineralization and provide clues for determining the mechanisms of myostracum formation and muscle-shell attachment.

Biomimetic synthesis of magnetic nanoparticles mediated by magnetosome proteins

Magnetotactic bacteria magnetosomes are magnetic nanoparticles enclosed by biofilms and arrange in the form of a chain. Magnetic nanoparticles formed by the biomineralization of magnetosome usually have regular shape, uniform particle size and high crystallinity, which have attracted extensive attention of researchers. The magnetosome membrane is composed of phospholipids and fatty acids, and the magnetosome membrane lipid vesicle acts as a nanoreactor which controls the precise synthesis of magnetic nanoparticles. A series of biomineralization proteins in the magnetosome membrane control the iron transport, redox reaction, nucleation and growth of the magnetic nanoparticles. At present, the specific biomineralization process of magnetosome is still unclear and the large-scale production of magnetosome is difficult, so the biomimetic synthesis of magnetosome has been initiated. In vivo studies have shown that magnetosome proteins of magnetotactic bacteria including Mms6, MamC, MmsF, MamG and MamD play an important role in regulating the size and morphology of the magnetosome, and they have been identified as the best candidates for the biomimetic synthesis of magnetosome. Biomimetic synthesis of magnetic nanoparticles mediated by recombinant magnetosome proteins such as Mms6, MamC and MmsF has been studied. These studies can not only help us better understand the biomineralization process of magnetosome, but also help us prepare high-quality magnetosome-like magnetic nanoparticles without the use of organic solvents, surfactants and high reaction temperature. This article mainly reviews the research progress of the biomimetic synthesis of magnetic nanoparticle mediated by several key magnetosome proteins and prospects its future development. Mms6 seems to mainly control over the growth kinetics of magnetic crystal by acting as an iron reservoir. MamC seems to preferably control the nucleation kinetics of magnetic crystals due to the ionotropic and template effects. MamP as an iron oxidase can support to form ferrihydrite which required for the formation of magnetite crystals in vivo . Although Mms6 is the most abundant magnetosome protein and there are the most researches on Mms6 mediated biomimetic synthesis of magnetic nanoparticles, the specific regulation mechanism of Mms6 is still unclear. In addition, the production cost of Mms6 is relatively high. These are all the shortcomings of Mms6 mediated biomimetic synthesis of magnetic nanoparticles. Therefore, in order to realize large scale biomimetic preparation of high-quality magnetic nanoparticles with controllable size and morphology, it is necessary to further clarify the regulation mechanism of Mms6 and develop cheaper and simpler methods to prepare Mms6 based functional polypeptides and additives. The biomineralization of magnetosome is a process regulated by a variety of mineralization proteins. Therefore, if several kinds of magnetosome proteins with different functions were used together to regulate the formation process of magnetic nanocrystal in the biomimetic synthesis methods, magnetic nanocrystals with more abundant morphology, size and surface crystal structure would be synthesized. Particularly, if MamP, MamE and other magnetosome proteins which control the redox reaction were used to assist the biomimetic synthesis of magnetic nanocrystals, the synthesis conditions might be further optimized, and the yield and performance of magnetic nanoparticles might be improved. In addition, constructing a micro lipid vesicle reactor which like the magnetosome membrane through the surface assembly of key magnetosome proteins and phospholipid molecules to control the biomimetic synthesis process is expected to introduce novel ideas and methods for the biomimetic synthesis of magnetic nanoparticles.

Investigating the ferric ion binding site of magnetite biomineralisation protein Mms6

The biomineralization protein Mms6 has been shown to be a major player in the formation of magnetic nanoparticles both within the magnetosomes of magnetotactic bacteria and as an additive in synthetic magnetite precipitation assays. Previous studies have highlighted the ferric iron binding capability of the protein and this activity is thought to be crucial to its mineralizing properties. To understand how this protein binds ferric ions we have prepared a series of single amino acid substitutions within the C-terminal binding region of Mms6 and have used a ferric binding assay to probe the binding site at the level of individual residues which has pinpointed the key residues of E44, E50 and R55 involved in Mms6 ferric binding. No aspartic residues bound ferric ions. A nanoplasmonic sensing experiment was used to investigate the unstable EER44, 50,55AAA triple mutant in comparison to native Mms6. This suggests a difference in interaction with iron ions between the two and potential changes to the surface precipitation of iron oxide when the pH is increased. All-atom simulations suggest that disruptive mutations do not fundamentally alter the conformational preferences of the ferric binding region. Instead, disruption of these residues appears to impede a sequence-specific motif in the C-terminus critical to ferric ion binding.

High strength brushite bioceramics obtained by selective regulation of crystal growth with chiral biomolecules

Chirality seems to play a key role in mineralization. Indeed, in biominerals, the biomolecules that guide the formation and organization of inorganic crystals and help construct materials with exceptional mechanical properties, are homochiral. Here, we show that addition of homochiral l-(+)-tartaric acid improved the mechanical properties of brushite bioceramics by decreasing their crystal size, following the classic Hall-Petch strengthening effect; d-(-)-tartaric acid had the opposite effect. Adding l-(+)-Tar increased both the compressive strength (26 MPa) and the fracture toughness (0.3 MPa m1/2) of brushite bioceramics, by 33% and 62%, respectively, compared to brushite bioceramics without additives. In addition, l-(+)-tartaric acid enabled the fabrication of cements with high powder-to-liquid ratios, reaching a compressive strength and fracture toughness as high as 32.2 MPa and 0.6 MPa m1/2, respectively, approximately 62% and 268% higher than that of brushite bioceramics prepared without additives, respectively. Characterization of brushite crystals from the macro- to the atomic-level revealed that this regulation is attributable to a stereochemical matching between l-(+)-tartaric acid and the chiral steps of brushite crystals, which results in inhibition of brushite crystallization. These findings provide insight into understanding the role of chirality in mineralization, and how to control the crystallographic structure of bioceramics to achieve high-performance mechanical properties. Statement of SignificanceCalcium-phosphate cements are promising bone repair materials. However, their suboptimal mechanical properties limit their clinical use. Natural biominerals have remarkable mechanical properties that are the result of controlled size, shape and organization of their inorganic crystals. This is achieved by biomineralization proteins that are homochiral, composed of l- amino acids. Despite the importance of chiral l-biomolecules in biominerals, using homochiral molecules to fabricate bone cements has not been studied yet. In this study, we showed that homochiral l-(+)-tartaric acid can regulate the crystal structure and improve the mechanical properties of a calcium-phosphate cement. Hence, these findings open the door for a new way of designing strong bone cement and highlight the importance of chirality in bioceramics.

In situ 3D visualization of biomineralization matrix proteins

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.

PI3K inhibition highlights new molecular interactions involved in the skeletogenesis of Paracentrotus lividus embryos

The sea urchin embryo develops a well-defined biomineralized endoskeleton, synthesized exclusively by the skeletogenic cells, supported by ectodermal cues for the correct skeleton patterning. The biomineralization process is tightly regulated via a hierarchical order of gene expression, including transcription and growth factors, biomineralization proteins. Recently, the role of kinases and intracellular signaling pathways in sea urchin skeletogenesis has been addressed, although the downstream components still remain unknown. In this study, we investigated the role of phosphatidylinositide 3-kinase (PI3K)-mediated signaling pathway in Paracentrotus lividus, to identify its genes/proteins targets. The effects of LY294002 (LY), a PI3K-specific inhibitor, were evaluated at morphological and molecular levels. Treatment with 40 μM LY from the blastula stage completely blocked skeleton deposition, which was reversed by wash out experiments. Besides, LY caused a slight delay in the tripartite gut development. Despite the skeleton absence, a few skeleton-specific proteins/mRNAs were regularly expressed and localized in LY-treated embryos, as shown for MSP130 and SM50 by immunofluorescence and in situ hybridization experiments. QPCR analyses showed that LY differently affected the expression of genes coding for other biomineralization proteins, transcription and growth factors. SM30 and carbonic anhydrase expression was severely downregulated, while almost all the transcription factors analyzed were upregulated. Based on the present results and in silico analyses, we propose an “interactomic” model simulating PI3K connections in P. lividus embryos. Our findings define a novel regulatory step in the embryonic skeletogenesis, and provide valuable molecular data for further studies on the role of PI3K signaling in invertebrate biomineralization.

Ubiquitin Designer Proteins as a New Additive Generation toward Controlling Crystallization.

Proteins controlling mineralization in vivo are diverse, suggesting that there are various ways by which mineralization can be directed in bioinspired approaches. While well-defined three-dimensional (3D) structures occur in biomineralization proteins, the design of synthetic, soluble, bioinspired macromolecules with specific, reproducible, and predictable 3D arrangements of mineral-interacting functions poses an ultimate challenge. Thus, the question of how certain arrangements of such functions on protein surfaces influence mineralization and in what ways specific alterations subsequently affect this process remains elusive. Here we used genetically engineered ubiquitin (Ub) proteins in order to overcome the limitations of generic bioinspired additive systems. By advancing existing protocols, we introduced an unnatural amino acid and subsequently mineral-interacting functions via selective-pressure incorporation and click chemistry, respectively, without affecting the Ub secondary structure. Indeed, as-obtained Ub with three phosphate functions at defined positions shows unique effects based on a yet-unmatched capability toward the stabilization of a film of a dense liquid mineral phase visible even with the naked eye and its transformation into amorphous nanoparticles and afterward crystals with complex shapes. We thereby demonstrate that Ub designer proteins pose a unique new generation of crystallization additives where the 3D arrangement of mineral-interacting functions can be designed at will, promising their future use for programmable, target-oriented mineralization control.

Composite Materials Design: Biomineralization Proteins and the Guided Assembly and Organization of Biomineral Nanoparticles.

There has been much discussion of the role of proteins in the calcium carbonate biomineralization process, particularly with regard to nucleation, amorphous stabilization/transformation, and polymorph selection. However, there has been little if any discussion of the potential role that proteins might play in another important process: the guided assembly and organization of mineral nanoparticles into higher-ordered structures such as mesocrystals. This review discusses particle attachment theory and recent evidence of mineral-associated proteins forming hydrogels that assemble and organize mineral clusters into crystalline phase. From this discussion we postulate a mechanism by which biomineralization protein hydrogel aggregation assists in mineral nanoparticle assembly and organization within calcium carbonate skeletal elements and discuss potentials ways for harnessing this process in materials design.

Preparation and Characterization of Biomimetic Hydroxyapatite Nanocrystals by Using Partially Hydrolyzed Keratin as Template Agent.

Hydroxyapatite (HA), a typical inorganic component of bone, is a widely utilized biomaterial for bone tissue repair and regeneration due to its excellent properties. Inspired by the recent findings on the important roles of protein in biomineralization and natural structure of fish scales, keratin was chosen as a template for modulating the assembly of HA nanocrystals. A series of HA nanocrystals with different sizes were synthesized by adjusting the concentration of partially hydrolyzed keratin. The structure and compositions of the prepared HA were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Raman spectrum, and Transmission electron microscopy (TEM). Results revealed that the size of the synthesized HA nanocrystals can be controlled by adjusting the concentration of partially hydrolyzed keratin. Specifically, the size of synthesized HA decreased from 63 ± 1.5 nm to 27 ± 0.9 nm with the increasing concentration of partially hydrolyzed keratin from 0 to 0.6g. In addition, in vitro cytocompatibility of synthesized HA nanocrystals were evaluated using the MG-63 cells.

Solid-State NMR and MD Study of the Structure of the Statherin Mutant SNa15 on Mineral Surfaces.

Elucidation of the structure and interactions of proteins at native mineral interfaces is key to understanding how biological systems regulate the formation of hard tissue structures. In addition, understanding how these same proteins interact with non-native mineral surfaces has important implications for the design of medical and dental implants, chromatographic supports, diagnostic tools, and a host of other applications. Here, we combine solid-state NMR spectroscopy, isotherm measurements, and molecular dynamics simulations to study how SNa15, a peptide derived from the hydroxyapatite (HAP) recognition domain of the biomineralization protein statherin, interacts with HAP, silica (SiO2), and titania (TiO2) mineral surfaces. Adsorption isotherms are used to characterize the binding affinity of SNa15 to HAP, SiO2, and TiO2. We also apply 1D 13C CP MAS, 1D 15N CP MAS, and 2D 13C-13C DARR experiments to SNa15 samples with uniformly 13C- and 15N-enriched residues to determine backbone and side-chain chemical shifts. Different computational tools, namely TALOS-N and molecular dynamics simulations, are used to deduce secondary structure from backbone and side-chain chemical shift data. Our results show that SNa15 adopts an α-helical conformation when adsorbed to HAP and TiO2, but the helix largely unravels upon adsorption to SiO2. Interactions with HAP are mediated in general by acidic and some basic amino acids, although the specific amino acids involved in direct surface interaction vary with surface. The integrated experimental and computational approach used in this study is able to provide high-resolution insights into adsorption of proteins on interfaces.

Crystallization of Pearl Biomineralization Protein in Microgravity Environments

Crystallization of Pearl Biomineralization Protein in Microgravity Environments

Biomineral proteins associated with the crystal growth of calcium carbonate

Biomineral proteins associated with the crystal growth of calcium carbonate

Activated Sludge Biomass as Magnetic Biosorbent

Background. Heavy metal pollution is a typical issue for a number of industries, and their removal is crucial for the environmental ecology. Biomass can be used as cheap and eco-friendly sorbent, for example excess activated sludge, because its utilization is a major expense in water treatment. Sorbents with magnetic properties can be extracted using fast and efficient magnetic separation method. Mechanism of biomineralization biogenic magnetic nanoparticles (BMN) is proved to be general for all three domains, so it is important to study what amount of activated sludge microorganisms are potential BMN producents. Objectives. The objective of this study is to determine potential BMN producents among microorganisms of activated sludge using methods of comparative genomics, to obtain magnetic biosorbent and investigate its efficiency in Fe 2+ removal from FeSO 4 solution (500 mg/L). Methods. For estimating homologies between biomineralization proteins of Μ agnetospirillum gryphiswaldense MSR-1 and microorga­nisms of activated sludge were used methods of pairwise and multiple genomic sequence alignment using free access tool BLAST (National Centre for Biotechnological Information, USA). For obtaining magnetic sorbent from activated sludge biomass high gradient magnetic separation was used. Results. Bioinformatic analysis showed, that among studied microorganisms of activated sludge, whose genomes are sequenced per 25% and more, all appeared to be potential BMN producers ( Stentor coeruleus , Bodo saltans , Sphaerotilus natans , Beggiatoa alba B18LD, Oscillatoria acuminata PCC 6304, Oscillatoria sp. PCC 10802, Oscillatoria nigro-viridis PCC 7112, Rivularia sp. PCC 7116, Anabaena sp. CRKS33, Anabaena sp. WA113, Anabaena sp. AL93, Anabaena sp. PCC7108, Anabaena MDT 14b, Thiothrix nivea DSM 5205, Duganella zoogloeoides ATCC 25935), 12 of them – potential producents of intracellular crystalline BMN. It was shown, that Fe 2+ removal efficiency isequal for non-separated activated sludge and both magnetic and non-magnetic fractions. Conclusions. Results show that excess sludge biomass can be used to obtain magnetic sorbent, which can be made without the use of extra magnetization with artificial magnetic nanoparticles. This allows extracting sorbent using methods of high gradient magnetic separation. Sorption results of Fe 2+ for magnetically labelled biosorbent on the base of activated sludge microorganisms experimentally proves equal efficiency of magnetically labelled, non-labelled fractions and biomass which was not exposed to HGMS and is higher than 97%.

Solid-State NMR Identification of Intermolecular Interactions in Amelogenin Bound to Hydroxyapatite

Biomineralization processes govern the formation of hierarchical hard tissues such as bone and teeth in living organisms, and mimicking these processes could lead to the design of new materials with specialized properties. However, such advances require structural characterization of the proteins guiding biomineral formation to understand and mimic their impact. In their “active” form, biomineralization proteins are bound to a solid surface, severely limiting our ability to use many conventional structure characterization techniques. Here, solid-state NMR spectroscopy was applied to study the intermolecular interactions of amelogenin, the most abundant protein present during the early stages of enamel formation, in self-assembled oligomers bound to hydroxyapatite. Intermolecular dipolar couplings were identified that support amelogenin dimer formation stabilized by residues toward the C-termini. These dipolar interactions were corroborated by molecular dynamics simulations. A β-sheet structure was identified in multiple regions of the protein, which is otherwise intrinsically disordered in the absence of hydroxyapatite. To our knowledge, this is the first intermolecular protein-protein interaction reported for a biomineralization protein, representing an advancement in understanding enamel development and a new general strategy toward investigating biomineralization proteins.

Bionformation Detection of Magnetic Nanoparticles Producers Among Iron- and Manganese-Oxidizing Bacteria

Background. In Ukraine, more than 17 % of underground water intakes are classified as unfunded in terms of iron content and 4.4 % in terms of manganese content. The increased content of these elements leads to a deterioration in the organoleptic properties of drinking water, the formation of precipitation and overgrowing of the water supply networks due to the development of iron-oxidizing bacteria. Therefore, it is important to study the magnetic properties of iron- and manganese-oxidizing bacteria for the possibility of their use in magnetic separation technologies in order to improve water treatment technologies with excess iron and manganese content.Objective. The aim of the paper is to search for potential producers of BMN among iron- and manganese-oxidizing bacteria and classify them by an internal structure (crystalline or amorphous) and BMN allocation (extracellular or intracellular), using comparative genomics methods.Methods. To evaluate the degree of similarity of BMN biomineralization proteins in MTB Magnetospirillum gryphiswaldense MSR-1 and in iron- and manganese-oxidizing bacteria, pair and multiple alignment methods using the free access program BLAST of the National Center for Biotechnological Information were used.Results. The bioinformatic analysis showed that among the 30 investigated iron- and manganese-oxidizing bacteria which belong to 5 genera (Gallionella, Siderocapsa, Sphaerotilus, Hyphomicrobium, Leptothrix), 28 microorganisms are potential producers of BMN.Conclusions. Conclusions are made about the prospects of further investigation of the influence of the magnetic field on the microorganisms of genera: Leptotrix, Sphaerotillus, Gallionella, Hyphomicrobium. An important direction for further experiments is the study of possible ways of conducting magnetic separation of iron- and manganese-oxidizing bacteria to prevent secondary contamination of drinking water and the formation of scales in pipelines. The use of the magnetic properties of the investigated microorganisms is useful for the development of ways to remove old scales within pipelines.

Noninvasive Microcomputerized X-ray Tomography Visualization of Mineralization Directed by Sea Urchin- and Nacre-Specific Proteins

The biomineralization process offers novel principles for crystal engineering and solid-state chemistry, but to achieve this we must first understand how organisms such as the mollusk and sea urchin craft skeletal elements such as the shell and embryonic spicule, respectively. In vitro studies of mollusk- and sea urchin-associated proteins reveal that these proteins form hydrogel particles that control the nucleation process, assemble mineral nanoparticles, and modify the surfaces and interiors of existing crystals. However, visualization of these processes is hampered by destructive and invasive methods such as those used for conventional scanning electron microscopy/transmission electron microscopy. In this report we detail the novel use of microcomputerized X-ray tomography (μCT) imaging to nondestructively investigate the in vitro calcium carbonate mineralization process in the presence of a recombinant sea urchin spicule matrix protein, rSpSM50, and a recombinant mollusk shell nacre protein, rPif97. Relative to the protein-free control scenario, both proteins generate calcite crystals that are consistent with the results obtained from previous studies, but with μCT we discovered new features; each protein generates a different number of mineral deposits, exhibits unique domains or motifs, and creates a stratification of mineral phases into layers. These results coincide with the known function of these proteins in vitro and in situ, and provide new information regarding biomineralization protein hydrogels and how they influence nucleation and crystal growth. We foresee that μCT imaging could, for appropriately sized systems, offer a bridge between in vitro experiments and in situ investigations of time-resolved nucleation and crystal growth phenomena in organisms, tissues, and synthetic materials.