Simona Fermani

Oriented Crystallization of Vaterite in Collagenous Matrices

The influence of high super- saturation on kinetic control and the importance of the polypeptide structure in the crystallization of calcium carbo- nate polymorphs were studied in cross- linked gelatin films containing high concentrations of the polypeptides poly-l-aspartate and poly-l-glutamate. Oriented crystallization of vaterite oc- curs in uniaxially deformed gelatin films containing poly-l-aspartate at concenta- tions greater than 100 mg per gram of gelatin. The fact that no orientation of the mineral phase was observed with entrapped poly-l-glutamate at the same concentrations suggests that the orient- ed crystallization is controlled by the b sheet structure assumed by poly-l-as- partate in the presence of calcium ions. These results indicate that local super- saturation in the microenvironment in which nucleation and growth occur plays an important role in controlling the deposition of vaterite in cross-linked gelatin films. However, collagen bundles and the ordered and oriented polypep- tide chains of poly-l-aspartate can con- tribute to the control of polymorphism by inducing the formation of a specific phase by epitaxial crystallization, as suggested by the preferentially oriented deposition of vaterite and aragonite. This is of potential significance in bio- mineralization processes and in materi- als science.

Protein crystallization on polymeric film surfaces

Abstract Polymeric films containing ionizable groups, such as sulfonated polystyrene, cross-linked gelatin films with adsorbed poly- l -lysine or entrapped poly- l -aspartate and silk fibroin with entrapped poly- l -lysine or poly- l -aspartate, have been tested as heterogeneous nucleant surfaces for proteins. Concanavalin A from jack bean and chicken egg-white lysozyme were used as models. It was found that the crystallization of concanavalin A by the vapor diffusion technique, is strongly influenced by the presence of ionizable groups on the film surface. Both the induction time and protein concentration necessary for the crystal nucleation decrease whereas the nucleation density increases on going from the reference siliconized cover slip to the uncharged polymeric surfaces and even more to the charged ones. Non-specific attractive and local interactions between the protein and the film surface might promote molecular collisions and the clustering with the due symmetry for the formation of the crystal nuclei. The results suggest that the studied polymeric film surfaces could be particularly useful for the crystallization of proteins from solutions at low starting concentration, thus using small quantities of protein, and for proteins with very long crystallization time.

Molecular mechanism of thioredoxin regulation in photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. Unlike lower photosynthetic organisms, which only contain A4–GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB–GAPDH depends on the redox state of a pair of cysteines of the CTE, which can form a disulfide bond under control of thioredoxin f, leading to specific inhibition of the NADPH-dependent activity. The tridimensional structure of A2B2–GAPDH from spinach chloroplasts, crystallized in the oxidized state, shows that each disulfide-containing CTE is docked into a deep cleft between a pair of A and B subunits. The structure of the CTE was derived from crystallographic data and computational modeling and confirmed by site-specific mutagenesis. Structural analysis of oxidized A2B2–GAPDH and chimeric mutant [A+CTE]4–GAPDH revealed that Arg-77, which is essential for coenzyme specificity and high NADPH-dependent activity, fails to interact with NADP in these kinetically inhibited GAPDH tetramers and is attracted instead by negative residues of oxidized CTE. Other subtle changes in catalytic domains and overall conformation of the tetramers were noticed in oxidized A2B2–GAPDH and [A+CTE]4–GAPDH, compared with fully active A4–GAPDH. The CTE is envisioned as a redox-sensitive regulatory domain that can force AB–GAPDH into a kinetically inhibited conformation under oxidizing conditions, which also occur during dark inactivation of the enzyme in vivo.

Protein crystallisation on chemically modified mica surfaces

Chemically modified mica sheets have been tested as heterogeneous nucleant surfaces for lysozyme, concanavalin A and thaumatin. Smooth mica surfaces with reduced hydrophilic properties and different density of ionisable groups have been prepared by a silanisation reaction using mixtures of n-propyltriethoxysilane and 3-aminopropyltriethoxysilane in different percentages starting from 0 to 100% of aminosilane. The crystallisation experiments were carried out with the hanging drop vapour diffusion technique. The results suggest that these mica surfaces act as heterogeneous nucleant agents, whose effectiveness is due to non-specific attractive and local interactions between charged residues of the protein and the ionisable groups on the mica surfaces.

Structure and morphology of synthetic magnesium calcite

The X-ray crystal structure refinements of two synthetic calcite single crystals containing 3.1 and 5.7 mol% magnesium were carried out. The structure of the second crystal was also refined at –80 °C. No significant structural difference from biogenic magnesium calcite was found. Magnesium within the crystals of calcite forms a solid solution over the compositional range investigated. The principal axes of vibration ellipsoids at –80 °C resemble very closely those of calcite suggesting that the large thermal parameters in magnesium calcite reflect increased thermal vibrations more than positional disorder. The crystals of magnesium calcite express new rhombohedral faces due to the interaction of magnesium with growing crystals. The role of magnesium in the morphology of single crystal and globular aggregates of magnesium calcite is discussed in view of its possible relevance in both biomineralization and crystal growth.

Crystallization of calcium carbonate salts into beta-chitin scaffold.

Composites of beta-chitin with calcium carbonate polymorphs were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. The three main polymorphs of calcium carbonate: aragonite, calcite and vaterite, were observed. Their location within the matrix is a function of the polymorph. The supersaturation inside the compartmentalized space in the chitin governs the location and polymorphism of the crystals.

Polymorphism and architectural crystal assembly of calcium carbonate in biologically inspired polymeric matrices

The control of the polymorphism and architectural crystal assembly of calcium carbonate minerals in gels formed by means of collagenous matrices with entrapped polypeptides is reported. It has been observed that the calcium carbonate polymorphic selectivity is related to the local supersaturation within the microenvironment where nucleation and growth occur. This crucial parameter is controlled in terms of the entrapped additive concentration and of the tailoring of the biopolymeric scaffold by mechanical deformation. Specific orientation effects and crystal aggregation of the mineral phases can be controlled either by the charged polypeptide with a beta structure or by the structural organization of the triple helical stretches in the collagenous matrix. This results in the growth and assembly of crystals into desired shapes and sizes by molecular recognition at a definite crystal face or by the control of the organic macromolecular microenvironment fit in the emerging area of biologically inspired approach to structured inorganic materials with appropriate physical and chemical properties.

Oriented crystallization of octacalcium phosphate into beta-chitin scaffold.

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.

Crystallization of proteins on functionalized surfaces

Functionalized mica sheets and polystyrene films exposing ionisable groups have been used as heterogeneous nucleating surfaces for model proteins. Surfaces with different densities of amino or sulfonated groups have been prepared. Crystallization trials were carried out using the hanging-drop vapour-diffusion method. The results show that using these surfaces the starting protein concentration necessary to form crystals is reduced. The effect of these surfaces on the crystallization process may be the consequence of electrostatic interactions between charged residues of proteins and ionisable groups on surfaces. These interactions can be attractive or repulsive, depending on the relative charge of the protein and the surface at the crystallization pH. Both phenomena can induce an increase of the local protein concentration on the surface or in its proximity, favouring nucleation. Moreover, a reduction of the waiting time (an estimation of the nucleation time) was also observed for some proteins, suggesting a surface-stabilization effect on crystal nuclei.

Keratin-lipid structural organization in the corneous layer of snake.

The shed epidermis (molt) of snakes comprises four distinct layers. The upper two layers, here considered as β‐layer, contain essentially β‐keratin. The following layer, known as mesos‐layer, is similar to the human stratum corneum, and is formed by thin cells surrounded by intercellular lipids. The latter layer mainly contains α‐keratin. In this study, the molecular assemblies of proteins and lipids contained in these layers have been analyzed in the scale of two species of snakes, the elapid Tiger snake (TS, Notechis scutatus) and the viperid Gabon viper (GV, Bitis gabonica). Scanning X‐ray micro‐diffraction, FTIR and Raman spectroscopies, thermal analysis, and scanning electron microscopy experiments confirm the presence of the three layers in the GV skin scale. Conversely, in the TS molt a typical α‐keratin layer appears to be absent. In the latter, experimental data suggest the presence of two domains similar to those found in the lipid intercellular matrix of stratum corneum. X‐ray diffraction data also allow to determine the relative orientation of keratins and lipids. The keratin fibrils are randomly oriented inside the layers parallel to the surface of scales while the lipids are organized in lamellar structures having aliphatic chains normal to the scale surface. The high ordered lipid organization in the mature mesos layer probably increases its effectiveness in limiting water‐loss.