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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.

In higher plants, NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant enzyme that exists in different isoenzymic forms. In Arabidopsis thaliana, three genes (Gdh1, Gdh2 and Gdh3) encode three different GDH subunits (beta, alpha and gamma) that randomly associate to form a complex array of homo- and heterohexamers. The modification of the GDH isoenzyme pattern and its regulation was studied during the development of A. thaliana in the gdh1, gdh2 single mutants and the gdh1-2 double mutant, with particular emphasis on GDH3. Investigations showed that the GDH3 isoenzyme could not be detected in closely related Arabidopsis species. The induction and regulation of GDH3 activity in the leaves and roots was investigated following nitrogen deprivation in the presence or absence of sucrose or kinetin. These experiments indicate that GDH3 is likely to play an important role during senescence and nutrient remobilization. (C) 2013 Elsevier Masson SAS. All rights reserved.

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.

We report the isolation of AtL1, a 249 bp non-LTR retrotransposon fragment from Arabidopsis thaliana by fingerprinting mRNAs extracted from A. thaliana plants, ecotype Columbia, in different heat stress conditions. Southern blot and PCR analysis suggested that AtL1 occurs as a single- or low-copy insert in the genome of A. thaliana ecotype Columbia. The presence of AtL1 in the genome of different Arabidopsis ecotypes was confirmed by PCR amplification and sequencing thus excluding all possible contamination. A preliminary scan of the AtL1 nucleotide sequence against the EMBL and NCBI databases revealed a high degree of similarity to a group of LINE type L1 retrotransposons of mammals and with a cDNA sequence of Artemisia annua. A phylogenetic analysis of LINE elements from animals and plants placed AtL1 and A. annua sequences in close proximity to some mammalian sequences but distant from the other plants LINE elements including those from Arabidopsis.

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic α-helix followed by a proline-rich segment that represents a ligand for SH3 domain-containing proteins. Here, we have determined using solution nuclear magnetic resonance spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the α2-peptide) comprising these two structural motifs, in association with dodecylphosphocholine (DPC) micelles. The structure was calculated using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The experimental studies were complemented by molecular dynamics simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in association with a DPC micelle in silico. The experimental and theoretical results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane association via the amphipathic α-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-containing proteins such as Fyn and contribute to our understanding of myelin architecture at the molecular level.

The interaction between 2',3'-cyclic nucleotide 3'-phosphodiesterase and guanine/adenine nucleotides was investigated. The binding of purine nucleotides to 2',3'-cyclic nucleotide 3'-phosphodiesterase was revealed by both direct and indirect methods. In fact, surface plasmon resonance experiments, triphosphatase activity measurements, and fluorescence experiments revealed that 2',3'-cyclic nucleotide 3'-phosphodiesterase binds purine nucleotide triphosphates with an affinity higher than that displayed for diphosphates; on the contrary, the affinity for both purine monophosphates and pyrimidine nucleotides was negligible. An interpretation of biological experimental data was achieved by a building of 2',3'-cyclic nucleotide 3'-phosphodiesterase N-terminal molecular model. The structural elements responsible for nucleotide binding were identified and potential complexes between the N-terminal domain of CNP-ase and nucleotide were analyzed by docking simulations. Therefore, our findings suggest new functional and structural property of the N-terminal domain of CNPase.

Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Ω-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.

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.

The blue-light sensitive protein YtvA from Bacillus subtilis is built of a photoactive, flavin-binding LOV (Light, Oxygen and Voltage) domain and a STAS domain with unknown function. Here we show that YtvA binds a fluorescent derivative of guanosine triphosphate (GTPTR) that can be displaced by both GTP or ATP. Unspecific NTP (N=G or A) binding is supported by the molecular model of YtvA-STAS. Blue-light activation of YtvA results in small and dark-reversible spectroscopic changes for GTPTR, suggesting that light-driven conformational changes are transmitted from the LOV core to the GTPTR binding site. These results support the idea that STAS domains may have a general NTP binding role and open a way to investigate the molecular functionality of YtvA-STAS.

The myelin basic protein (MBP) is the second most abundant protein in the myelin sheath of the central nervous system and is believed to be important for the compactness and integrity of the membrane. We investigated the mechanism of the interaction of lipid-free MBP with phospholipid monolayers at the air/water interface; in particular, we studied the process of MBP adsorption onto monolayers made up either of neutral dipalmitoylphosphatidylcholine (DPPC) or of negatively charged dipalmitoylphosphatidylserine (DPPS) monolayers. They are natural constituents of the myelin membrane, and sharing an identical hydrophobic chain, they differ only in headgroup composition. The MBP−lipid interaction is investigated for the first time by means of nullellipsometric measurements, monitoring in real time the effect of adsorbed molecules in the insoluble monolayer at different monolayer conditions, such as surface pressure and molecular area. The different behavior of monolayer thickness and surface pressure confirme...

A prokaryotic protein, YtvA from Bacillus subtilis, was found to possess a light, oxygen, voltage (LOV) domain sharing high homology with the photoactive, flavin mononucleotide (FMN)-binding LOV domains of phototropins (phot), blue-light photoreceptors for phototropism in higher plants. Computer-based three-dimensional modeling suggests that YtvA-LOV binds FMN in a similar pocket as phot-LOVs. Recombinant YtvA indeed exhibits the same spectroscopical features and blue-light-induced photochemistry as phot-LOVs, with the reversible formation of a blue-shifted photoproduct, assigned to an FMN-cysteine thiol adduct (Thio383). By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio383, Phi(Thio) = 0.49, and the enthalpy change, DeltaH(Thio) = 135 kJ/mol, with respect to the parent state. The formation of Thio383 is accompanied by a considerable volume contraction, DeltaV(Thio) = -13.5 ml/mol. Similar to phot-LOVs, Thio383 is formed from the decay of a red-shifted transient species, T650, within 2 micros. In both YtvA and free FMN, this transient has an enthalpy content of approximately 200 kJ/mol, and its formation is accompanied by a small contraction, DeltaV(T) approximately -1.5 ml/mol, supporting the assignment of T650 to the FMN triplet state, as suggested by spectroscopical evidences. These are the first studies indicating that phototropin-related, blue-light receptors may exist also in prokaryotes, besides constituting a steadily growing family in plants.

NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant and ubiquitous enzyme that may exist in different isoenzymic forms. Variation in the composition of the GDH isoenzyme pattern is observed during plant development and specific cell, tissue and organ localization of the different isoforms have been reported. However, the mechanisms involved in the regulation of the isoenzymatic pattern are still obscure. Regulation may be exerted at several levels, i.e. at the level of transcription and translation of the relevant genes, but also when the enzyme is assembled to originate the catalytically active form of the protein. In Arabidopsis thaliana, three genes (GDH1, GDH2 and GDH3) encode three different GDH subunits (β, α and γ) that randomly associate to form a complex array of homo- and hetero-hexamers. In order to asses if the different Arabidopsis GDH isoforms may display different structural properties we have investigated their thermal stability. In particular the stability of GDH1 and GDH3 isoenzymes was studied using site-directed mutagenesis in a heterologous yeast expression system. It was established that the carboxyl terminus of the GDH subunit is involved in the stabilization of the oligomeric structure of the enzyme.

Myelin basic protein (MBP) efficiently bound Dilauroyl- l α-phosphatidic acid (DLPA) monolayers at the air water interface. Langmuir–Blodgett (LB) films were prepared from these monolayers and the hydrophilic surface of the upper layer was inspected by atomic force microscopy (AFM) in a water solution. The topography images of nominally four-layer films of DLPA in the absence of MBP revealed regions of different thickness corresponding to different numbers of lipid bimolecular layers. This morphology is characteristic of those lipid films which reorganise spontaneously when kept under an aqueous solution. The DLPA films containing MBP did not reorganise; their thicknesses were uniform and surfaces were usually covered by disordered clusters of protein molecules. Quasi-ordered arrays of smaller particles were observed in films with small amounts of bound protein. The surface charge densities of DLPA and DLPA-MPA films were investigated performing force-distance measurements by using tips modified with surface carboxyl groups. The curves obtained on pure lipid films showed repulsive forces well described in terms of double layer forces. Repulsive forces were not observed in the presence of a uniform MBP overlayer indicating that MBP neutralised most of the lipid charge.

A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G M1 , and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3–4 nm along them.

Static and dynamic spectroscopic properties of the tryptophanil emission in conjunction with circular dichroism (CD) spectroscopy and molecular dynamics are used to investigate the interactions of the neuropeptide neuromedin B (NMB) and the membrane-permeable delta sleep-inducing peptide (DSIP) with the membrane lipid phase. Our data indicate that in solution both peptides exist in energetically equivalent conformations, whereas in the presence of the membrane specific conformational states are stabilized. By changing from the aqueous to the lipid phase, the static and the dynamic fluorescence properties of the NMB's tryptophan residue are clearly affected: the fluorescence steady-state spectrum as well as the resolved fluorescence decay-associated spectra (DAS) are shifted to the blue with a significant increase of the fluorescence intensity of the second lifetime component (tau 2-DAS). On the other hand, in the lipid environment the same parameters of DSIP are negligibly affected as compared to the aqueous buffer. The CD and molecular dynamics analyses are consistent with these results and indicate that, while NMB assumes a helix-like conformation with the tryptophan residue in the apolar surface, DSIP adopts a globule-like structure with the indole ring that is surface-exposed. As previously found for neuromedin C (Polverini, E., Neyroz, P., Fariselli, P., Casadio, R., and Masotti, L., Biochem. Biophys. Res. Commun. 214, 663-668, 1995), for NMB the stabilized "lipophilic" structure also may favor the correct peptide-receptor contact and recognition. For DSIP, the lipid-stabilized conformation does not support an amphiphilic structure-driven peptide-membrane interaction and suggests a hydrophobicity-driven diffusion across the bilayer.

p13suc1 acts in the fission yeast cell division cycle as a component of p34cdc2. In the present work, structural information contained in the intrinsic fluorescence of p13suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13suc1 is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained with p13suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13suc1 is best described by three discrete lifetimes of 0.6 ns (τ1), 2.9 ns (τ2), and 6.1 ns (τ3), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-τ2 (λmax = 332 nm) and DAS-τ3 (λmax = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (kq(τ2)≫kq(τ3)) are resolved for each lifetime. An interpretation in terms of specific tryptophan residue (or protein conformer)-lifetime assignments is presented. The decay of the fluorescence anisotropy of native p13suc1 is best described by a double exponential decay. The longer correlation time recovered (9 ns ≤ Φ2 ≤ 15ns) can be associated with the rotational motion of the protein as a whole and a Stokes radius of 21.2 A has been calculated for p13suc1. Anisotropy measurements obtained as a function of temperature indicate that, in solution, the protein exists exclusively as a prolate monomer. In 1 mM zinc, changes of the anisotropy decay parameters are compatible with subunits oligomerization.

Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/β scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

The stability and functionality of GCC-bOBP, a monomeric triple mutant of bovine odorant binding protein, was investigated, in the presence of denaturant and in acidic pH conditions, by both protein and 1-aminoanthracene ligand fluorescence measurements, and compared to that of both bovine and porcine wild type homologues. Complete reversibility of unfolding was observed, though refolding was characterized by hysteresis. Molecular dynamics simulations, performed to detect possible structural changes of the monomeric scaffold related to the presence of the ligand, pointed out the stability of the β-barrel lipocalin scaffold.

Myelin basic protein (MBP) is a highly post-translationally modified, multifunctional structural component of central nervous system myelin, adhering to phospholipid membranes and assembling cytoskeletal proteins, and has previously been shown to bind SH3 domains in vitro and tether them to a membrane surface [Polverini, E., et al. (2008) Biochemistry 47, 267-282]. Since molecular modeling shows that the Fyn-SH3 domain has a negative surface charge density even after binding the MBP ligand, we have investigated the influence of negative membrane surface charge and the effects of post-translational modifications to MBP on the interaction of the Fyn-SH3 domain with membrane-associated MBP. Using a sedimentation assay with multilamellar vesicles consisting of neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol (PI), we demonstrate that increasing the negative surface charge of the membrane by increasing the proportion of PI reduces the amount of Fyn-SH3 domain that binds to membrane-associated MBP, due to electrostatic repulsion. When one of the phosphoinositides, PI(4)P or PI(4,5)P(2) was substituted for PI in equal proportion, none of the Fyn-SH3 domain bound to MBP under the conditions that were used. Post-translational modifications of MBP which reduced its net positive charge, i.e., phosphorylation or arginine deimination, increased the degree of repulsion of Fyn-SH3 from the membrane surface, an effect further modulated by the lipid charge. This study suggests that changes in membrane negative surface charge due to protein or lipid modifications, which could occur during cell signaling, can regulate the binding of the Fyn-SH3 domain to membrane-associated MBP and thus could regulate the activity of Fyn at the oligodendrocyte membrane surface.

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic R-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin ) PexD ) Abl ) Fyn ) c-Src ) Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin ) c-Src > PexD ) Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH‚‚‚﷿ interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP- peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation -﷿ interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues.

The interaction between 2',3'-cyclic nucleotide 3'-phosphodiesterase and guanine/adenine nucleotides was investigated. The binding of purine nucleotides to 2',3'-cyclic nucleotide 3'-phosphodiesterase was revealed by both direct and indirect methods. In fact, surface plasmon resonance experiments, triphosphatase activity measurements, and fluorescence experiments revealed that 2',3'-cyclic nucleotide 3'-phosphodiesterase binds purine nucleotide triphosphates with an affinity higher than that displayed for diphosphates; on the contrary, the affinity for both purine monophosphates and pyrimidine nucleotides was negligible. An interpretation of biological experimental data was achieved by a building of 2',3'-cyclic nucleotide 3'-phosphodiesterase N-terminal molecular model. The structural elements responsible for nucleotide binding were identified and potential complexes between the N-terminal domain of CNP-ase and nucleotide were analyzed by docking simulations. Therefore, our findings suggest new functional and structural property of the N-terminal domain of CNPase.

Objective: To verify whether a synthetic therapeutic killer decapeptide (KP), a functional mimotope of a yeast killer toxin with wide-spectrum microbicidal activity, inclusive of AIDS-related opportunistic micro-organ isms, through interaction with P-glucan receptors, which has been found to possess sequence homology with critical segments in gp160 V1/V2 and V3 loops, may also be inhibiting HIV-1 replication. Methods: Primary peripheral blood mononuclear cells (PBMCs) cultures established from HIV-1-infected patients were treated with KP in comparison with zidovudine and supernatants and cells were harvested for analysis of HIV RNA and proviral contents, respectively. Virus production in exogenous in-vitro PBMCs infection with lymphocytotropic and monocytotropic HIV-1 strains was also assessed in presence of KP by enzyme-linked immunosorbent assay HIV p24 gag antigen detection. The binding affinity of KP to CD4, CCR5 and CXCR4 was evaluated on CD4-CCR5 or CD4-CXCR4 transfected astroglioma cell lines. Results: KP was shown to be devoid of cytotoxicity on PBMCs and to inhibit HIV-1 replication in PBMCs of a patient in the acute phase of infection. The antiretroviral activity of KP, which proved to be more potent than zidovudine at micromolar concentrations, is abolished by beta 1,3-glucan but not by beta 1,6-glucan. Down-regulation of CCR5 co-receptor, and/or physical block of the gp120-receptor interaction are possible mechanisms of KP activity. Conclusion: KP appears to be the first anti body-derived short peptide displaying an inhibitory activity against HIV-1 and related opportunistic micro-organisms by different mechanisms of action. (c) 2006 Lippincott Williams & Wilkins.

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands β-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.

The classic 18.5 kDa isoform of murine myelin basic protein (mMBP) has been shown to bind calmodulin (CaM) strongly and specifically in vitro. Here, we have used site-directed spin labelling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to map more precisely the sites of interaction of recombinant mMBP (rmMBP) with CaM. On the basis of these and previous experimental data, and the predictions of CaM-binding motifs using the Calmodulin Target Database ( http://calcium.uhnres.utoronto.ca ), three main segments of MBP were suggested for the interaction. The first site is located at the C-terminus; the second one lies in the central portion of the protein and forms an amphipathic α-helix in reconstituted myelin-mimetic systems; the third is quite close to the N-terminus. The murine Golli-MBP isoform J37 has also been shown to bind CaM in vitro, and an interaction site was predicted in the N-terminal Golli-specific portion of the protein. From these four segments, we selected peptide fragments of 12–14 residues in length, chosen on the bases of their amphipathicity and CaM-target characteristics. We modelled each of these peptides as α-helices, and performed docking simulations to investigate their interactions with the CaM peptide-binding tunnel. Different yet almost equally favourable CaM-binding modes were found for each of them. The experimental SDSL/EPR and theoretical modelling results were in good agreement, and supported the conjecture that there are several plausible CaM-binding sites in MBP, that could be induced into an α-helical conformation by their interaction with CaM and account for strong immobilisation of spin-labeled residues in all three segments. Phosphorylation and deimination were also emulated and simulated for known sites of MBP post-translational modification. The results obtained confirmed the appropriate utilisation of simple residue substitutions to mimic the natural modifications, and demonstrated molecular mechanisms by which MBP–CaM interactions could be modulated in vivo.

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having alpha-helix and beta-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results.

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 x 4.2 x 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis. The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.

The 18.5 kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5 kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5 kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.

The P2 protein is a neuritogenic, small basic protein present in PNS myelin. It belongs to the family of the cytoplasmic lipid-binding proteins and can be incorporated in lipidic bilayers. P2 has been purified and crystallized only in the lipid-free form. Here we show that the P2 protein can be purified with bound lipids by applying to PNS myelin the same procedure that as used to purify lipid-bound myelin basic protein from CNS myelin. SDS-PAGE showed a single band of 16.5 kDa, and TLC showed the presence of most of the myelin lipids associated with the protein. Lipid-bound P2 revealed different circular dichroism spectra from the corresponding lipid-free form, indicating that lipids influence P2 conformation.