Marcelo Ceolín

Comparative Structural and Chemical Studies of Ferritin Cores with Gradual Removal of their Iron Contents

Transmission Electron Microscopy (TEM), X-ray Absorption Near Edge Spectroscopy (XANES), Electron Energy-Loss Spectroscopy (EELS), Small-Angle X-ray Scattering (SAXS), and SQUID magnetic studies were performed in a batch of horse spleen ferritins from which iron had been gradually removed, yielding samples containing 2200, 1200, 500, and 200 iron atoms. Taken together, findings obtained demonstrate that the ferritin iron core consists of a polyphasic structure (ferrihydrite, magnetite, hematite) and that the proportion of phases is modified by iron removal. Thus, the relative amount of magnetite in ferritin containing 2200 to 200 iron atoms rose steadily from approximately 20% to approximately 70% whereas the percentage of ferrihydrite fell from approximately 60% to approximately 20%. These results indicate a ferrihydrite-magnetite core-shell structure. It was also found that the magnetite in the ferritin iron core is not a source of free toxic ferrous iron, as previously believed. Therefore, the presence of magnetite in the ferritin cores of patients with Alzheimers disease is not a cause of their increased brain iron(II) concentration.

Electrochemical Sensing Platform Based on Polyelectrolyte–Surfactant Supramolecular Assemblies Incorporating Carbon Nanotubes

The characterization and application of a polyelectrolyte-surfactant supramolecular assembly formed by poly(allylamine) and dodecyl sulfate (PA-DS) on a screen-printed graphite electrode for the preparation of electrochemical sensing platforms are presented. The system was characterized by X-ray reflectometry (XRR) and grazing-incidence small-angle X-ray scattering (GISAXS) and tested with four benchmark electrochemical probes undergoing different electron-transfer mechanisms on carbon: ferrocyanide, hexaammineruthenium, ascorbic acid, and dopamine. The polyelectrolyte acts as a scaffold favoring the incorporation of the ferrocyanide, an ion oppositely charged to poly(allylamine). Also, its ability to incorporate carbon nanotubes (CNT) is presented. The composite material PA-DS-CNT is able to electrocatalyze the oxidation of dopamine, allowing its detection at micromolar levels in the presence of 100 times higher concentrations of ascorbate and it is shown to be stable, while XRR and GISAXS results confirm a lamellar structure with well-defined domains, not perturbed by the presence of the CNT. The dispersion is easily prepared in aqueous solution and could facilitate the processing of the CNT with an efficient loading and yielding a more robust carbon-based material for sensing applications.

Redox-active concanavalin A: synthesis, characterization, and recognition-driven assembly of interfacial architectures for bioelectronic applications.

The convergence of chemistry, biology, and materials science has paved the way to the emergence of hybrid nanobuilding blocks that incorporate the highly selective recognition properties of biomolecules, with the tailorable functional capabilities of inorganic molecules. In this work, we describe for the first time the decoration of concanavalin A (Con A), a protein with the ability to recognize sugars and form glycoconjugates, with Os(II) redox-active complexes. This strategy enabled the construction of electroactive biosupramolecular materials whose redox potentials could be easily modulated through the facile molecular modification of the electroactive inorganic complexes. Small-angle X-ray scattering (SAXS), steady-state fluorescence, surface plasmon resonance (SPR) spectroscopy, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and differential-pulsed (DPV) and cyclic voltammetry (CV) were used to characterize the structural and functional features of the synthesized biohybrid building blocks as well as their respective supramolecular assemblies built up on gold electrodes. By harnessing the electroactive and carbohydrate-recognition properties of these tailor-made biohybrid building blocks, we were able to integrate glucose oxidase (GOx) onto gold electrodes via sugar-lectin interactions. The redox activity of the Os-modified Con A interlayer allowed the electronic connection between the multilayered GOx assemblies and the metal electrode as evidenced by the well-defined bioelectrocatalytic response exhibited by the biomolecular assemblies in the presence of the glucose in solution. We consider that this approach based on the spontaneous formation of redox-active biosupramolecular assemblies driven by recognition processes can be of practical relevance for the facile design of biosensors, as well as for the construction of new multifunctional bioelectrochemical systems.

A bioinspired approach to the synthesis of bimetallic CoNi nanoparticles.

Bimetallic CoNi nanoparticles have been prepared within the apoferritin cavity. The protein shell controls size, prevents aggregation, and makes nanoparticles water-soluble. The CoNi series prepared in this way were structurally and magnetically characterized, the resulting magnetic properties varying accordingly with composition (Co(75)/Ni(25), Co(50)/Ni(50), Co(25)/Ni(75)). Co and Ni metals were associated in each nanoparticle, as demonstrated by high-angle annular dark field scanning electron microscopy and electron energy loss spectroscopy (EELS). After intentional oxidation, the CoNi nanoparticles were characterized by EELS, X-ray absorption near edge structure (XANES), and SQUID measurements to evaluate the importance of the oxidation on magnetic properties.

Structure and stability of the neurotoxin PV2 from the eggs of the apple snail Pomacea canaliculata

There is little information on the egg proteins of gastropod mollusks. Here we focus on PV2, a novel neurotoxin from snail eggs, studying its size, shape, structure, and stability, using small angle X-ray scattering (SAXS), absorption and fluorescence spectroscopy, circular dichroism, electron microscopy and partial proteolysis. Results indicate that PV2 is a compact and well folded oligomer of 130x44 A. It is an octamer of four 98 kDa heterodimers composed of 67 and 31 kDa subunits. Subunits are held together by disulfide bonds. Dimers are assembled into native PV2 by non-covalent forces. The larger subunit is more susceptible to proteolysis, indicating it is less compactly folded and/or more exposed. Quenching of tryptophan fluorescence showed a single class of tryptophyl side chains occluded in hydrophobic regions. Native structure shows loss of secondary structure (alpha+beta) at 6 M urea or 60-70 degrees C; the effects on the quaternary structure suggest an unfolding without disassembling of the protein. The 3D model of PV2 presented here is the first for an egg proteinaceous neurotoxin in animals.

Engineering a compact non-native state of intestinal fatty acid-binding protein

The last three C-terminal residues (129-131) of intestinal fatty acid-binding protein (IFABP) participate in four main-chain hydrogen bonds and two electrostatic interactions to sequentially distant backbone and side-chain atoms. To assess if these interactions are involved in the final adjustment of the tertiary structure during folding, we engineered an IFABP variant truncated at residue 128. An additional mutation, Trp-6-->Phe, was introduced to simplify the conformational analysis by optical methods. Although the changes were limited to a small region of the protein surface, they resulted in an IFABP with altered secondary and tertiary structure. Truncated IFABP retains some cooperativity, is monomeric, highly compact, and has the molecular dimensions and shape of the native protein. Our results indicated that residues 129-131 are part of a crucial conformational determinant in which several long-range interactions, essential for the acquisition of the native state, are established. This work suggests that carefully controlled truncation can populate equilibrium non-native states under physiological conditions. These non-native states hold a great promise as experimental models for protein folding.

Grafting a new metal ligand in the cocatalytic site of B. cereus metallo‐β‐lactamase: Structural flexibility without loss of activity

Metallo‐β‐lactamases are zinc enzymes able to hydrolyze the four‐membered ring of β‐lactam antibiotics, representing one of the latest generations of β‐lactamases. These enzymes belong to the zinc metallo‐hydrolase family of the β‐lactamase fold. Enzymes belonging to this family have a bimetallic active site whose structure varies among different members by point substitutions of the metal ligands. In this work, we have grafted new metal ligands into the metal binding site of BcII from Bacillus cereus that mimic the ligands present in other members of this superfamily. We have characterized spectroscopically and modeled the structure of the redesigned sites, which differ substantially from the wild‐type enzyme. Despite the changes introduced in the active site, the mutant enzymes retain almost full activity. These results shed some light on the possible evolutionary origin of these metalloenzymes.

Self-assembly of PBzMA-b-PDMAEMA diblock copolymer films at the air–water interface and deposition on solid substrates via Langmuir–Blodgett transfer

The aggregation behavior and morphological characteristics of amphiphilic block copolymer polybenzyl methacrylate-block-poly(dimethylamino)ethyl methacrylate (PBzMA-b-PDMAEMA) at the air–water interface were investigated through surface pressure measurements, atomic force microscopy (AFM) imaging, electrochemical measurements and X-ray reflectivity. Ionization of PDMAEMA blocks significantly affects the isotherms of the surface films at the air–water interface and, consequently, originates different morphologies of Langmuir–Blodgett films obtained under different experimental conditions. At low pH the PDMAEMA blocks are fully protonated and therefore dissolved in the aqueous subphase. Under this condition, the effects of the solubility and electrostatic repulsion of submerged PDMAEMA chains prevail over hydrophobic interactions and the Langmuir film exhibits low surface pressure at large molecular area values. Upon compression, the isotherm shows a pseudoplateau corresponding to the “pancake-to-brush” transition followed by an increase in surface pressure at smaller MMA values. These results were correlated with the morphological features of Langmuir–Blodgett films transferred onto silicon substrates, where dispersed dot-like domains that gradually transformed into an island-like structure, followed by further percolation into a continent-like morphology, were observed through AFM imaging. On the other hand, at high pH the isotherm is more expanded and the film exhibits higher surface pressure at relatively high MMA values due to the strong repulsive interactions between interface-confined hydrophobic aggregates constituted of PBzMA cores and neutral PDMAEMA shells. In this case, the AFM results show a structural evolution from circular and quasi-hexagonally packed micelles, followed by a worm-like structure that collapses into a homogeneous film. Furthermore, the study of the copolymer behavior under different subphase ionic strength conditions confirmed the critical role of electrostatic interactions in determining the characteristics of the isotherm. We could also demonstrate that our system follows accurately the scaling relationship for surface pressure of annealed brushes. Complementary studies performed by means of X-ray reflectivity were carried out to probe the buried interfacial structure of the diblock copolymer film, corroborating that the organization of both blocks on the silicon substrates is strongly dependent on the pH conditions of the subphase during the LB transfer.

Agglutinating activity and structural characterization of scalarin, the major egg protein of the snail Pomacea scalaris (d'Orbigny, 1832).

Apple snail perivitellins are emerging as ecologically important reproductive proteins. To elucidate if the protective functions of the egg proteins of Pomacea canaliculata (Caenogastropoda, Ampullariidae), involved in embryo defenses, are present in other Pomacea species we studied scalarin (PsSC), the major perivitellin of Pomacea scalaris. Using small angle X-ray scattering, fluorescence and absorption spectroscopy and biochemical methods, we analyzed PsSC structural stability, agglutinating activity, sugar specificity and protease resistance. PsSC aggluttinated rabbit, and, to a lesser extent, human B and A erythrocytes independently of divalent metals Ca2+ and Mg2+ were strongly inhibited by galactosamine and glucosamine. The protein was structurally stable between pH 2.0 to 10.0, though agglutination occurred only between pH 4.0 to 8.0 (maximum activity at pH 7.0). The agglutinating activity was conserved up to 60°C and completely lost above 80°C, in agreement with the structural thermal stability of the protein (up to 60°C). PsSC was able to withstand in vitro gastrointestinal digestion, and showed no trypsin inhibition activity. The presence of lectin activity has been reported in eggs of other Pomacea snails, but here we link for the first time, this activity to an apple snail multifunctional perivitellin. This novel role for a snail egg storage protein is different from closely related P.canaliculata defensive proteins.

Thermoreversible formation and negative thermal expansion of supramacromolecular assemblies of unimolecular micelles in solution.

We report the thermoreversible formation of superstructural assemblies of unimolecular micelles in solution displaying negative thermal expansion behaviour.