Marco Chino

Femtosecond UV-laser pulses to unveil protein–protein interactions in living cells

A hallmark to decipher bioprocesses is to characterize protein–protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.

Novel human bioactive peptides identified in apolipoprotein B : evaluation of their therapeutic potential

Graphical abstract Figure. No Caption available. ABSTRACT Host defence peptides (HDPs) are short, cationic amphipathic peptides that play a key role in the response to infection and inflammation in all complex life forms. It is increasingly emerging that HDPs generally have a modest direct activity against a broad range of microorganisms, and that their anti‐infective properties are mainly due to their ability to modulate the immune response. Here, we report the recombinant production and characterization of two novel HDPs identified in human Apolipoprotein B (residues 887–922) by using a bioinformatics method recently developed by our group. We focused our attention on two variants of the identified HDP, here named r(P)ApoBL and r(P)ApoBS, 38‐ and 26‐residue long, respectively. Both HDPs were found to be endowed with a broad‐spectrum antimicrobial activity while they show neither toxic nor haemolytic effects towards eukaryotic cells. Interestingly, both HDPs were found to display a significant anti‐biofilm activity, and to act in synergy with either commonly used antibiotics or EDTA. The latter was selected for its ability to affect bacterial outer membrane permeability, and to sensitize bacteria to several antibiotics. Circular dichroism analyses showed that SDS, TFE, and LPS significantly alter r(P)ApoBL conformation, whereas slighter or no significant effects were detected in the case of r(P)ApoBS peptide. Interestingly, both ApoB derived peptides were found to elicit anti‐inflammatory effects, being able to mitigate the production of pro‐inflammatory interleukin‐6 and nitric oxide in LPS induced murine macrophages. It should also be emphasized that r(P)ApoBL peptide was found to play a role in human keratinocytes wound closure in vitro. Altogether, these findings open interesting perspectives on the therapeutic use of the herein identified HDPs.

A De Novo Heterodimeric Due Ferri Protein Minimizes the Release of Reactive Intermediates in Dioxygen‐Dependent Oxidation

Metalloproteins utilize O2 as an oxidant, and they often achieve a 4-electron reduction without H2 O2 or oxygen radical release. Several proteins have been designed to catalyze one or two-electron oxidative chemistry, but the de novo design of a protein that catalyzes the net 4-electron reduction of O2 has not been reported yet. We report the construction of a diiron-binding four-helix bundle, made up of two different covalently linked α2 monomers, through click chemistry. Surprisingly, the prototype protein, DF-C1, showed a large divergence in its reactivity from earlier DFs (DF: due ferri, two iron). DFs release the quinone imine and free H2 O2 in the oxidation of 4-aminophenol in the presence of O2 , whereas FeIII -DF-C1 sequesters the quinone imine into the active site, and catalyzes inside the scaffold an oxidative coupling between oxidized and reduced 4-aminophenol. The asymmetry of the scaffold allowed a fine-engineering of the substrate binding pocket, that ensures selectivity.

Enhancement of Peroxidase Activity in Artificial Mimochrome VI Catalysts through Rational Design

Rational design provides an attractive strategy to tune and control the reactivity of bioinspired catalysts. Although there has been considerable progress in the design of heme oxidase mimetics with active‐site environments of ever‐growing complexity and catalytic efficiency, their stability during turnover is still an open challenge. Herein, we show that the simple incorporation of two 2‐aminoisobutyric acids into an artificial peptide‐based peroxidase results in a new catalyst (FeIII‐MC6*a) with higher resistance against oxidative damage and higher catalytic efficiency. The turnover number of this catalyst is twice as high as that of its predecessor. These results point out the protective role exerted by the peptide matrix and pave the way to the synthesis of robust bioinspired catalysts.

Oxidation catalysis by iron and manganese porphyrins within enzyme-like cages

Inspired by natural heme‐proteins, scientists have attempted for decades to design efficient and selective metalloporphyrin‐based oxidation catalysts. Starting from the pioneering work on small molecule mimics in the late 1970s, we have assisted to a tremendous progress in designing cages of different nature and complexity, able to accommodate metalloporphyrins. With the intent of tuning and controlling their reactivity, more and more sophisticated and diverse environments are continuously exploited. In this review, we will survey the current state of art in oxidation catalysis using iron‐ and manganese‐porphyrins housed within designed or engineered protein cages. We will also examine the innovative metal‐organic framework (MOF) systems, exploited to achieving an enzyme‐like environment around the metalloporphyrin cofactor.

Unveiling the structure of a novel artificial heme-enzyme with peroxidase-like activity: A theoretical investigation

Fe(III)‐Mimochrome VI (MC6) is a recently reported artificial heme‐peptide conjugate system with a high peroxidase‐like activity. By design, its structure features a five‐coordinated Fe(III)‐deuteroporphyrin active site, embedded in a compact α‐helix–heme–α‐helix “sandwich” motif. Up to now, no detailed MC6 structural characterization is available. In this work we propose a theoretical investigation based on molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) optimizations, aimed to shed light on several Fe(III)‐MC6 structural features and to validate the de novo designed fold. Key structural elements were analyzed to achieve indirect insight relevant to understand Fe(III)‐MC6 catalytic performances in solution. Extensive MD simulations showed a partial stability of the “sandwich” fold in water solution. The smaller peptide chain bonded to the heme revealed a high conformational freedom, which promoted the exposition of the heme distal side to the solvent. Regarding the accessibility of water molecules, even in Fe(III)‐MC6 “closed” structure the heme cavity appeared hydrated, suggesting an easy accessibility by exogenous ligands. Fe(III)‐MC6 structure in both high and low spin states was then further characterized through hybrid QM/MM optimizations. In particular, an accurate description of the active site structure was obtained, allowing a direct comparison of Fe(III)‐MC6 coordination environment with that observed in the Horseradish Peroxidase crystal structures. Our results suggest a structural similarity between Fe(III)‐MC6 and the natural enzyme. This study supports the interpretation of data from experimental Fe(III)‐MC6 structural and functional characterization and the rational design of new artificial mimics with improved catalytic performances.

Exploring the role of unnatural amino acids in antimicrobial peptides

Cationic antimicrobial peptides (CAMPs) are a promising alternative to treat multidrug-resistant bacteria, which have developed resistance to all the commonly used antimicrobial, and therefore represent a serious threat to human health. One of the major drawbacks of CAMPs is their sensitivity to proteases, which drastically limits their half-life. Here we describe the design and synthesis of three nine-residue CAMPs, which showed high stability in serum and broad spectrum antimicrobial activity. As for all peptides a very low selectivity between bacterial and eukaryotic cells was observed, we performed a detailed biophysical characterization of the interaction of one of these peptides with liposomes mimicking bacterial and eukaryotic membranes. Our results show a surface binding on the DPPC/DPPG vesicles, coupled with lipid domain formation, and, above a threshold concentration, a deep insertion into the bilayer hydrophobic core. On the contrary, mainly surface binding of the peptide on the DPPC bilayer was observed. These observed differences in the peptide interaction with the two model membranes suggest a divergence in the mechanisms responsible for the antimicrobial activity and for the observed high toxicity toward mammalian cell lines. These results could represent an important contribution to unravel some open and unresolved issues in the development of synthetic CAMPs.

Artificial Heme Enzymes for the Construction of Gold-Based Biomaterials

Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.

Spectroscopic and metal binding properties of a de novo metalloprotein binding a tetrazinc cluster: Chino et al.

De novo design provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen‐rich environments remains in its infancy. In previous work, we described the design of homotetrameric four‐helix bundles that bind tetra‐Zn2+ clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal‐binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the corresponding apo‐proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn2+ in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in the apo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster.