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Mr4511 from Methylobacterium radiotolerans is a photoreceptor of the light, oxygen voltage (LOV) family, binding flavin mononucleotide (FMN) as a chromophore. It exhibits the prototypical LOV photocycle, with the reversible formation of an FMN-Cys71 adduct via fast decay of the FMN triplet state. Mr4511 has high potential as a photosensitiser for singlet oxygen (SO) upon mutation of C71. Mr4511-C71S shows a triplet lifetime (τ T) of several hundreds of microseconds, ensuring efficient energy transfer to dioxygen to form SO. In this work, we have explored the potential diffusion pathways for dioxygen within Mr4511 using molecular dynamics (MD) simulations. The structural model of wild-type (wt) Mr4511 showed a dimeric structure stabilised by a strong leucine zipper at the two C-terminal helical ends. We then introduced in silico the C71S mutation and analysed transient and persistent oxygen channels. MD simulations indicate that the chromophore binding site is highly accessible to dioxygen. Mutations that might favour SO generation were designed based on their position with respect to FMN and the oxygen channels. In particular, the C71S-Y61T and C71S-Y61S variants showed an increased diffusion and persistence of oxygen molecules inside the binding cavity.

The aim of this study was to investigate the action spectrum of two urea derivatives, the 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and the 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU). In order to evaluate a possible adjuvant activity on cytokinins the compounds alone or in the simultaneous presence of different cytokinins were assayed either on in vitro typical cytokinin-related bioassays, or on in planta interaction with cytokinin signal transduction pathway. The compounds ability to activate the cytokinin receptor CRE1/AHK4 was studied either by a heterologous bacterial assay or by a competitive binding assay and docking simulations were performed with the crystal structure of the same receptor. Then, owing to their chemical structure which resembles that of urea-type cytokinins, the ability of 5- and 6-BDPU to inhibit the activity of cytokinin oxidase/dehydrogenase of Zea mays (ZmCKX1) was investigated and docking simulations were performed as well. Accordingly to the experimental results, we speculate that BDPUs could show a dual activity: the blocking of the conformational re-adaption of CRE1/AHK4 receptor maintaining the cytokinin inside its binding pocket, thus possibly enhancing its kinase action; the inhibition of cytokinin oxidase/dehydrogenase activity thus possibly preventing its cleavage of natural cytokinins with isoprenoid side chain. Graphic abstract

In this work we exploited time-resolved photoacoustics (PA) and molecular dynamics (MD) simulations to investigate the function of a conserved phenylalanine residue in blue sensing (BL) LOV domains. The LOV photocycle involves reversible formation of a photoproduct (LOV390) where the flavin mononucleotide (FMN) chromophore is covalently bound to a cysteine. LOV390 thermally returns to the dark adapted state (LOV447) with a lifetime τrec (s-to-h). In the LOV domain of Bacillus subtilis BsYtvA, the conserved F46 is one of the few residues undergoing a pronounced light-driven conformational change. PA and spectroscopic data show that in the YtvA variants F46A and F46Y light-induced structural changes are much smaller than those in the wild type (wt) protein, τrec is strongly accelerated and the energy content of LOV390 is lower for F46Y. MD simulations for each variant in the LOV447 and LOV390 states revealed an overall very stable structure of the BsYtvA-LOV domain. The largest variations emerged for the conserved HB network that includes FMN, Q123 (the "flipping" glutamine of LOV domains), and the conserved N104 and N94, with strong dependence on the presence of water. The lateral chain of Q123 in wt-LOV447 can adopt three alternative conformations, and movements act in concert with F46 flexibility. In LOV390, Q123 remains instead fixed in the orientation adopted in the crystal structure. Interestingly, in F46A, Q123 is locked in a LOV447-like conformation (pseudo-dark-adapted state), in both LOV447 and LOV390. In LOV447 of F46Y the tyrosine hydroxyl group fixes a water molecule, which induces a Q123 conformation similar to wt-LOV390, i.e. a pseudo-photoproduct state. These pseudo-dark-adapted and photoproduct-like conformations of the Q123 sidechain may account for the strong acceleration of the photocycle in the two variants. Given the importance of the "flipping" glutamine in light-to-signal propagation in LOV proteins, the results presented here underscore a crucial structural and functional role of the conserved F46. MD results also indicate that F46 is not directly engaged in permeability of the FMN pocket, but is involved in solvent ordering and the formation of water bridges.

Collagen dehydration is an unavoidable damaging process that causes the lack of fibers' physical properties and it is usually irreversible. However, the identification of low hydration conditions that permit a recovering of initial collagen features after a rehydration treatment is particularly of interest. Monitoring structural changes by means of MD simulations, we investigated the hydration-dehydration-rehydration cycle of two microfibril models built on different fragments of the sequence of rat tail collagen type I. The microfibrils have different hydropathic features, to investigate the influence of amino acid composition on the whole process. We showed that with low hydration at a level corresponding to the first shell, microfibril gains in compactness and tubularity. Crucially, some water molecules remain trapped inside the fibrils, allowing, by rehydrating, a recovery of the initial collagen structural features. Water rearranges in cluster around the protein, and its first layer is more anchored to the surface. However, these changes in distribution and mobility in low hydration conditions get back with rehydration.

In this work we exploited time-resolved photoacoustics (PA) and molecular dynamics (MD) simulations to investigate the function of a conserved phenylalanine residue in blue sensing (BL) LOV domains. The LOV photocycle involves reversible formation of a photoproduct (LOV390) where the flavin mononucleotide (FMN) chromophore is covalently bound to a cysteine. LOV390 thermally returns to the dark adapted state (LOV447) with a lifetime τrec (s-to-h). In the LOV domain of Bacillus subtilis BsYtvA, the conserved F46 is one of the few residues undergoing a pronounced light-driven conformational change. PA and spectroscopic data show that in the YtvA variants F46A and F46Y light-induced structural changes are much smaller than those in the wild type (wt) protein, τrec is strongly accelerated and the energy content of LOV390 is lower for F46Y. MD simulations for each variant in the LOV447 and LOV390 states revealed an overall very stable structure of the BsYtvA-LOV domain. The largest variations emerged for the conserved HB network that includes FMN, Q123 (the "flipping" glutamine of LOV domains), and the conserved N104 and N94, with strong dependence on the presence of water. The lateral chain of Q123 in wt-LOV447 can adopt three alternative conformations, and movements act in concert with F46 flexibility. In LOV390, Q123 remains instead fixed in the orientation adopted in the crystal structure. Interestingly, in F46A, Q123 is locked in a LOV447-like conformation (pseudo-dark-adapted state), in both LOV447 and LOV390. In LOV447 of F46Y the tyrosine hydroxyl group fixes a water molecule, which induces a Q123 conformation similar to wt-LOV390, i.e. a pseudo-photoproduct state. These pseudo-dark-adapted and photoproduct-like conformations of the Q123 sidechain may account for the strong acceleration of the photocycle in the two variants. Given the importance of the "flipping" glutamine in light-to-signal propagation in LOV proteins, the results presented here underscore a crucial structural and functional role of the conserved F46. MD results also indicate that F46 is not directly engaged in permeability of the FMN pocket, but is involved in solvent ordering and the formation of water bridges.

Self-assembly of rat tail collagen type I was investigated by means of turbidity measurements and molecular dynamics simulations. Turbidity curves collected at different pH values show that the rate of aggregation was not linear in dependence from pH, with the fastest kinetics at pH 5.0 and the lowest at neutral pH. MD simulations were carried out on two regions with different hydropathicity, monitoring the aggregation of up to four staggered tropocollagen fragments at different ionic strength. At physiological conditions, association of lowly charged regions occurs more easily than for highly charged ones, the latter seeming to aggregate in a sequential way. The first contacts indicate for both regions that the driving force is hydrophobic, the electrostatic contribution becoming relevant at short distance. The direct inter-tropocollagen H-bonds confirm that fibrillogenesis is driven by loss of surface water from the monomers and involves in large percentage hydroxyproline residues. Low ionic strength dynamics leads to the formation of incorrect assemblies, driven by not shielded pairwise charge interactions.

Four Cellular Retinol-binding Proteins (CRBP 1, 2, 3, 4) are encoded in the human genome. CRBP 1 and 2, sharing a 56% amino acid sequence identity, exhibit the highest binding affinities for retinol. Previous NMR studies provided some insights into the mechanism of retinol uptake, but details of such mechanism remain to be elucidated. Herein, the results of molecular dynamics simulations for the uptake of retinol by CRBP 1 and 2 are consistent with the presence of two different retinol entry points, both involving the 'cap region' (α-helices I and II and neighboring loops). We observed that a hydrophobic patch at the surface of the 'portal region' (α-helix II, CD and EF loops) of CRBP 1 attracts retinol, which accesses the binding cavity through an opening generated by the concerted movements of Arg58 and Phe57, present in the CD loop. In CRBP 2 a different distribution of the surface residues of the 'cap region' allows retinol to access the binding cavity by sinking in a hydrophobic matrix between the two α-helices. Polar interactions mainly affect retinol movements inside the β-barrel cavities of both CRBPs. The interaction energy profiles are in agreement with the different behavior of the two protein systems.

Naja atra subsp. atra cardiotoxin 1 (CTX-1), produced by Chinese cobra snakes, belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. Using as template mainly the tip and the subsequent β-strand of the first "finger" of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-3, and its variants NCP-3a and NCP-3b, have shown the best antimicrobial activity, together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50-6.3 μg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families.

Four cellular retinol-binding protein (CRBP) types (CRBP1,2,3,4) are encoded in the human genome. Here, we report on X-ray analyses of human apo- and holo-CRBP1, showing nearly identical structures, at variance with the results of a recent study on the same proteins containing a His-Tag, which appears to be responsible for a destabilizing effect on the apoprotein. The analysis of crystallographic B-factors for our structures indicates that the putative portal region, in particular α-helix-II, along with Arg58 and the E-F loop, is the most flexible part of both apo- and holoprotein, consistent with its role in ligand uptake and release. Fluorometric titrations of wild type and mutant forms of apo-CRBP1, coupled with X-ray analyses, provided insight into structural and molecular determinants for the interaction of retinol with CRBP1. An approximately stoichiometric binding of retinol to wild type apo-CRBP1 (Kd∼4.5nM), significantly lower binding affinity for both mutants Q108L (Kd∼65nM) and K40L (Kd∼70nM) and very low binding affinity for the double mutant Q108L/K40L (Kd∼250nM) were determined, respectively. Overall, our data indicate that the extensive apolar interactions between the ligand and hydrophobic residues lining the retinol binding cavity are sufficient to keep it in its position bound to CRBP1. However, polar interactions of the retinol hydroxyl end group with Gln108 and Lys40 play a key role to induce a high binding affinity and specificity for the interaction.

A series of quinoline-2-carboxaldehyde thiosemicarbazones and their copper(II) and nickel(II) complexes were synthesized and characterized. In all complexes the ligands are in the E configuration with respect to the imino bond and behave as terdentate. The copper(II) complexes form square planar derivatives with one molecule of terdentate ligand and chloride ion. A further non-coordinated chloride ion compensates the overall charge. Nickel(II) ions form instead octahedral complexes with two ligands for each metal ion, independently from the stoichiometric metal:ligand ratio used in the synthesis. Ligands and complexes were tested for their antiproliferative properties on histiocytic lymphoma cell line U937. Copper(II) derivatives are systematically more active than the ligands and the nickel complexes. All copper derivatives result in inhibiting topoisomerase IIa in vitro. Computational methods were used to propose a model to explain the different extent of inhibition presented by these compounds. The positive charge of the dissociated form of the copper complexes may play a key role in their action.