Similarly to enzymes, functionalized gold nanoparticles efficiently catalyze chemical reactions, hence the term nanozymes. Herein, we present our results showing how surface‐passivated gold nanoparticles behave as synthetic nanonucleases, able to cleave pBR322 plasmid DNA with the highest efficiency reported so far for catalysts based on a single metal ion mechanism. Experimental and computational data indicate that we have been successful in creating a catalytic site precisely mimicking that suggested for natural metallonucleases relying on a single metal ion for their activity. It comprises one Zn(II) ion to which a phosphate diester of DNA is coordinated. Importantly, as in nucleic acids‐processing enzymes, a positively charged arginine plays a key role by assisting with transition state stabilization and by reducing the pK_a of the nucleophilic alcohol of a serine. Our results also show how designing a catalyst for a model substrate (bis‐p‐nitrophenylphosphate) may provide wrong indications as for its efficiency when it is tested against the real target (plasmid DNA).

A series of 2-nm gold nanoparticles passivated with different thiols all featuring at least one triazacyclonanone-Zn(II) complex and different flanking units (a second Zn(II) complex, a triethyleneoxymethyl derivative or a guanidinium of arginine of a peptide) were prepared and studied for their efficiency in the cleavage of the RNA-model substrate 2-hydroxypropyl-p-nitrophenyl phosphate. The source of catalysis for each of them was elucidated from the kinetic analysis (Michaelis–Menten profiles, pH dependence and kinetic isotope effect). The data indicated that two different mechanisms were operative: One involving two Zn(II) complexes and the other one involving a single Zn(II) complex and a flanking guanidinium cation. The mechanism based on a dinuclear catalytic site appeared more efficient than the one based on the cooperativity between a metal complex and a guanidinium.

Similarly to enzymes, functionalized gold nanoparticles efficiently catalyze chemical reactions, hence the term nanozymes. Herein, we present our results showing how surface‐passivated gold nanoparticles behave as synthetic nanonucleases, able to cleave pBR322 plasmid DNA with the highest efficiency reported so far for catalysts based on a single metal ion mechanism. Experimental and computational data indicate that we have been successful in creating a catalytic site precisely mimicking that suggested for natural metallonucleases relying on a single metal ion for their activity. It comprises one Zn(II) ion to which a phosphate diester of DNA is coordinated. Importantly, as in nucleic acids‐processing enzymes, a positively charged arginine plays a key role by assisting with transition state stabilization and by reducing the pK_a of the nucleophilic alcohol of a serine. Our results also show how designing a catalyst for a model substrate (bis‐p‐nitrophenylphosphate) may provide wrong indications as for its efficiency when it is tested against the real target (plasmid DNA).

In-vivo studies on wound healing have shown that small metal oxide nanoparticles (NPs), such as titania, ferria, and alumina, promote wound closure, likely by enhancing blood coagulation and via activation of the contact system. However, titania differs from the others as it supports the regrowth of normal skin tissue without scarring. This unique effect was hypothesised to result from its ability to suppress inflammation, possibly by interfering with the complement system. E x-vivo whole blood experiments within the present study confirm that titania NPs significantly dampen the immune response and surface plasmon resonance (SPR) biosensor analysis confirms that the nanoparticle binds with nanomolar affinity to complement C3, a central protein in the human innate immune system. To elucidate the molecular basis of this effect, we developed a novel cryo-electron microscopy (cryo-EM) methodology capable of resolving the challenging interface between metal NPs and proteins. This approach revealed that TiO□ binds with high specificity to a single domain on C3. The NP restricts the conformational dynamics of C3 and thus blocks the initiation of the complement cascade. These structural and biophysical insights support the biochemical findings and previous observations within the contact system. Our method advancement demonstrates that cryo-EM can uncover physiologically relevant interactions between proteins and nanomaterials at high resolution and offers a strong foundation for others investigating metal-biological interactions.

Interactions between peptides based on a region in the zinc finger translocation associated (ZFTA) protein and the Kelch domain of Kelch‐like protein 20 (KLHL20 ^Kelch ) have been characterised by biosensor analysis, supported by AlphaFold2‐based structure predictions of peptides bound to the protein. Residues critical for the interaction were identified. The analysis showed that all peptides exhibited relatively weak and complex interactions with KLHL20 ^Kelch . The original ZFTA peptide had a much higher affinity for KLHL20 ^Kelch than for the Kelch domain of KLHL12 (KLHL12 ^Kelch ), indicating a specificity for KLHL20 ^Kelch . The estimated K _D ^app of 35 µM was like that for a 21‐mer peptide derived from death‐associated protein kinase 1, a known KLHL20 substrate. Removal of flexible C‐terminal residues generated a 12‐mer, predicted to form a stable helix. This reduced the affinity 100‐fold. Removal of N‐terminal residues resulted in a 10‐mer predicted to be flexible, which had a similar affinity as the original 16‐mer. The similar affinities for peptides representing different regions of ZFTA suggest that the recognition is feature specific rather than sequence specific. The interaction mechanism reflects “fuzzy binding”, consistent with the role of KLHL20 as an adaptor protein in the ubiquitination of disordered protein substrates by Cullin‐3 E3 ubiquitin ligase.

Interactions between peptides based on a region in the zinc finger translocation associated (ZFTA) protein and the Kelch domain of Kelch-like protein 20 (KLHL20^Kelch) have been characterised by biosensor analysis, supported by AlphaFold2-based structure predictions of peptides bound to the protein. Residues critical for the interaction were identified. The analysis showed that all peptides exhibited relatively weak and complex interactions with KLHL20^Kelch. The original ZFTA peptide had a much higher affinity for KLHL20^Kelch than for the Kelch domain of KLHL12 (KLHL12^Kelch), indicating a specificity for KLHL20^Kelch. The estimated K_D ^app of 35 µM was like that for a 21-mer peptide derived from death-associated protein kinase 1, a known KLHL20 substrate. Removal of flexible C-terminal residues generated a 12-mer, predicted to form a stable helix. This reduced the affinity 100-fold. Removal of N-terminal residues resulted in a 10-mer predicted to be flexible, which had a similar affinity as the original 16-mer. The similar affinities for peptides representing different regions of ZFTA suggest that the recognition is feature specific rather than sequence specific. The interaction mechanism reflects "fuzzy binding", consistent with the role of KLHL20 as an adaptor protein in the ubiquitination of disordered protein substrates by Cullin-3 E3 ubiquitin ligase.