Giarita Ferraro

Self‐assembling proteins are widely used in bionanotechnology due to their biocompatibility, non‐toxicity, structural stability, and easy functionalization. Ferritins (Fts) are particularly attractive systems, as they assemble into highly ordered nanocages with a hollow internal cavity. Their assembly and disassembly can be modulated by various protocols, such as changes in pH or the use of denaturing agents, enabling their application in molecular encapsulation and drug delivery. However, the efficiency of encapsulation remains challenging, often resulting in low or heterogeneous loading and incomplete protein recovery. Here, we load peptides into the core of the human H‐chain ferritin ( hHFt ) by genetically fusing them to the C‐terminus of the Ft subunit, a region facing the inner cavity. As model peptides, we used fragments derived from the SARS ‐ CoV ‐2 Spike protein sharing similar physicochemical properties but different charges. The structure of the functionalized nanocages was assessed using techniques like transmission electron microscopy, native gel electrophoresis, and X‐ray crystallography. High‐resolution structural analysis revealed interesting conformational changes in the hHFt BC ‐loop associated with C‐terminal functionalization, while the overall nanocage architecture is preserved. Additionally, NMR measurements of the transverse relaxation ( T ₂ ) of a target peptide demonstrated that the nanocages do not expose the fused peptides under native conditions, but upon disassembly, can efficiently expose them, demonstrating that C‐terminal genetic functionalization of ferritin nanocages is a robust and effective approach for nanobiotechnological applications.

This study presents an integrated surface diagnostic investigation and a diagnostic-driven restoration of the marble sculpture Real Infante Carlo Tito di Borbone (1775), attributed to Giuseppe Sanmartino and preserved at the Royal Palace of Caserta (Italy). Despite the artistic relevance of the sculptor, scientific investigations of his works remain limited. The aim of this work is to characterize surface coatings, identify alteration processes, and support conservation strategies through a multi-analytical approach. The results reveal that the sculpture is composed of calcitic marble covered by an altered organic surface film. Spectroscopic and chromatographic data indicate the presence of lipidic compounds consistent with natural wax, likely associated with previous conservation treatments. An epoxy-based adhesive was also identified, suggesting past restoration interventions. Microbiological analyses detected fungal and bacterial species potentially involved in surface alteration processes. The integration of analytical data enabled the development of a targeted restoration strategy. Cleaning procedures were defined based on the chemical nature of the surface coatings, while structurally stable previous restorations were preserved. A biocide treatment was applied to mitigate biological colonization. This study highlights the importance of surface-oriented diagnostics in understanding coating materials and guiding conservation interventions, providing a methodological framework applicable to marble artworks.

Thioredoxin reductase from Burkholderia cenocepacia (Bc‐TrxR) is a recognized intracellular target of antibacterial gold(I) compounds, yet the molecular basis of enzyme inhibition remains unclear. Here, we report an integrated structural, biophysical, and biochemical investigation of recombinant Bc‐TrxR and its interaction with three prototypical gold(I) agents: auranofin ( AF ) and two trimethylphosphine‐thiolate derivatives ( Au1 and Au2 ), previously identified as potent enzyme inhibitors. The enzyme was expressed, purified, and its crystal structure solved at 2.52 Å resolution, revealing a homodimeric architecture closely resembling that of the Escherichia coli enzyme. Each subunit contains FAD‐ and NADPH‐binding domains and a catalytic Cys–Cys motif representing a plausible coordination site for gold fragments. High‐resolution ESI‐MS provided direct molecular evidence for the formation of defined gold‐protein adducts, with up to two Au(I) centres bound per subunit. The observed mass shifts are consistent with selective coordination of gold fragments to catalytically relevant cysteine residues, effectively blocking redox turnover. Together, crystallographic and mass spectrometric results disclose the molecular mechanism of Bc‐TrxR inhibition by phosphine‐thiolate gold(I) complexes and establish a structural framework for the rational design of next‐generation antibacterial metallodrugs.

The employment of metal-based compounds in medicine is an ever-changing topic. Following cisplatin discovery, last decades have seen a huge activity designing new metallodrugs with improved pharmacological profiles able to overcome issues related to administration of cisplatin and its derivatives. Despite promising features, metallodrugs entrance into clinical practice is hampered by a series of deficiencies including poor solubility and general toxicity, always coupled with lack of selectivity. Problems associated with the use of conventional drug therapy could be addressed through the development of carrier biomaterials able to ensure drug safe and targeted delivery to specific sites. Proteins have been widely explored for this purpose and ferritin, a protein with a hollow cage structure, represents the ideal candidate. This mini-review aims to summarize the studies investigating the use of ferritin-based formulations for the delivery of metal compounds of medical interest highlighting strength points and weaknesses of this potential nanocarrier.

The interaction of vanadium compounds of pharmaceutical interest with metal-transport proteins like human serum transferrin (hTF) is poorly understood. Direct structural evidence identifying vanadium binding sites on hTF is still lacking. Here, the X-ray structure of the adduct formed when the potential drug [V^IVO(acac)₂], with acac = acetylacetonato, reacts with human serum transferrin with Fe³⁺ bound at the C-lobe only (Fe_C-hTF) has been solved and compared with new structures of Fe_C-hTF used as controls. Structural analysis revealed the presence of a [V^V₂O₆]^2– anion that can be described as a divanadate(V) anion, [V^V₂O₇]^4–, that has one oxygen replaced by the phenolate oxygen of Tyr188. The two vanadium centers adopt tetrahedral geometry, consistent with V^V behavior. The binding does not alter the overall conformation of Fe_C-hTF that retains the open conformation of the N-lobe and the closed conformation of the C-lobe, remaining able to be recognized by the transferrin receptor.

Upon reaction of dirhodium tetraacetate ([Rh₂(μ-O₂CCH₃)₄]) and some [Rh₂(μ-O₂CCH₃)₄] derivatives with proteins, dimeric Rh-Rh units (diRh) or monometallic moieties can bind the side chains of His, Cys, Met, Asp, Asn, Arg and Lys, and the C-terminal carboxylate. However, structural data on the interaction between the diRh center and Cys and Met side chains within the protein environment are still missing. Here, we report the X-ray structure of the adduct that [Rh₂(μ-O₂CCH₃)₄] forms with C-phycocyanin from Galdiera phlegrea at 2.17 Å resolution. Twelve diRh binding sites were found on the protein structure, two for each (αβ) unit. Dimetallic fragments were observed close to the side chains of Met30 of β-chains and of Cys73 of α-chains. To the best of our knowledge, the results provide the first unambiguous crystallographic observation of the diRh center binding to Met and Cys protein residues. DiRh binding does not alter overall protein structure and stability. This result will help in the design of new dirhodium-based artificial metalloenzymes.

Cs₂[VV2O₄(malato)₂]·2H₂O can undergo transformation into a variety of V-containing species which can interact with proteins through multiple binding modes and stabilization mechanisms.

Paddlewheel dirhodium complexes are cytotoxic compounds that are also used as catalysts and in the formation of Rh-based artificial metalloenzymes. Low-temperature structures of adducts formed by the model protein hen egg white lysozyme (HEWL) with dirhodium tetraacetate ([Rh₂(μ-O₂CCH₃)₄]) when crystals of the protein were treated with the metal compound at 20 °C demonstrated that [Rh₂(μ-O₂CCH₃)₄] in part breaks down upon reaction with HEWL; dimeric Rh-Rh units bind the side chains of Asp18 and the C-terminal carboxylate, and monometallic fragments coordinate the side chains of Arg14 and His15 in 20% ethylene glycol, 0.100 M sodium acetate at pH 4.5 and 0.600 M sodium nitrate, while dimeric Rh-Rh units bind the side chains of Asn93 and Lys96, the C-terminal carboxylate and Asp101, with monometallic fragments that bind the side chains of Lys33 and His15 in 0.010 M HEPES pH 7.5 and 2.00 M sodium formate. To verify whether the binding of this metallodrug to proteins also occurs at body temperature, crystals of HEWL were grown in 0.010 M HEPES pH 7.5 and 2.00 M sodium formate at 37 °C and soaked with [Rh₂(μ-O₂CCH₃)₄] at the same temperature. X-ray diffraction data collected on these crystals at 37 °C demonstrate that [Rh₂(μ-O₂CCH₃)₄] reacts with proteins at body temperature. The structures of the Rh/HEWL adduct formed at 20 °C (obtained from data collected at 100 K) and at 37 °C under the same experimental conditions are very similar, with metal binding sites that are conserved. However, metal-containing fragment occupancy is higher in the structure obtained at 37 °C, suggesting a role of temperature in defining the protein metalation process.

Ferritin (Ft) is a protein with a peculiar three-dimensional architecture. It is characterized by a hollow cage structure and is responsible for iron storage and detoxification in almost all living organisms. It has attracted the interest of the scientific community thanks to its appealing features, such as its nano size, thermal and pH stability, ease of functionalization, and low cost for large-scale production. Together with high storage capacity, these properties qualify Ft as a promising nanocarrier for the development of delivery systems for numerous types of biologically active molecules. In this paper, we introduce the basic structural and functional aspects of the protein, and summarize the methods employed to load bioactive molecules within the ferritin nanocage.