Search

This work is based on previous evidence showing that chemotactic sequence of the urokinase receptor (uPAR88-92) drives angiogenesis in vitro and in vivo in a protease-independent manner, and that the peptide Ac-Arg-Glu-Arg-Phe-NH2 (RERF) prevents both uPAR88–92- and VEGF-induced angiogenesis. New N-acetylated and C-amidated peptide analogues containing α-methyl α-amino acids were designed and synthesized to optimize the biochemical properties for therapeutic applications. Among these, Ac-L-Arg-Aib-L-Arg-D-Cα(Me)Phe-NH2, named UPARANT, adopts in solution a turned conformation similar to that found for RERF, is stable to sterilization in 3 mg/mL sealed vials in autoclave for 20 minutes at 120°C, is stable in blood, and displays a long-time resistance to enzymatic proteolysis. UPARANT competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the formyl-peptide receptor, inhibits VEGF-directed endothelial cell migration, and prevents cytoskeletal organization and αvβ3 activation in endothelial cells exposed to VEGF. In vitro, UPARANT inhibits VEGF-dependent tube formation of endothelial cells at a 100× lower concentration than RERF. In vivo, UPARANT reduces to the basal level VEGF-dependent capillary sprouts originating from the host vessels that invaded Matrigel sponges implanted in mice, and completely prevents neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Both excellent stability and potency position UPARANT as a promising new therapeutic agent for the control of diseases fueled by excessive angiogenesis, such as cancer and inflammation. Mol Cancer Ther; 13(5); 1092–104. ©2014 AACR.

PDF file - 234K, Table S1 - Stability in physiological solution of UPARANT. Table S2 - Blood Stability of UPARANT. Table S3. Proton Chemical Shifts, 3JNH-alphaCH Coupling Constants of UPARANT at 298 K. Table S4. Average backbone torsion angles of RERF as obtained from RMD simulation in vacuo at 300 K. Figure S1 - 1H NMR spectra and selected regions of the ROESY spectra of UPARANT.

Video Description from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

Supplementary Data to Figure 3 from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

RERF Conformational Preferenc from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

Video from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

Dattabase Information from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O 2 -dependent oxidation of catechols to o -quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.

Indole oxidation represents a real challenge for selectivity because it typically leads to complex mixtures of products even when highly selective enzymes are used. Here, we describe the catalytic potential of an artificial enzyme, Mn-Mimochrome VI*a (Mn-MC6*a), in promoting indole oxidation. This mini-enzyme contains a manganese-deuteroporphyrin active site within a scaffold of two synthetic small peptides covalently linked to porphyrin. Mn-MC6*a promotes the selective oxidation of indole at its C3 position, leading to a 3-oxindole derivative (2-TFE-3-oxindole) with unprecedented product selectivity (86% at pH 8.5) compared to native or artificial heme-enzymes. We also suggest a possible reaction mechanism based on the effect of pH, cosolvent, and indole substitution on the reaction outcome. These studies demonstrate that Mn-MC6*a is an excellent artificial peroxygenase for the production of 3-oxindole-containing compounds as key building blocks for the synthesis of fine chemicals.

Artificial metalloenzymes as catalysts are promising candidates for their use in different technologies, such as bioremediation, biomass transformation, or biosensing. Despite this, their practical exploitation is still at an early stage. Immobilized natural enzymes have been proposed to enhance their applicability. Immobilization may offer several advantages: (i) catalyst reuse; (ii) easy separation of the enzyme from the reaction medium; (iii) better tolerance to harsh temperature and pH conditions. Here, we report an easy immobilization procedure of an artificial peroxidase on different surfaces, by means of click chemistry. FeMC6*a, a recently developed peroxidase mimic, has been functionalized with a pegylated aza-dibenzocyclooctyne to afford a "clickable" biocatalyst, namely FeMC6*a-PEG4@DBCO, which easily reacts with azide-functionalized molecules and/or nanomaterials to afford functional bioconjugates. The clicked biocatalyst retains its structural and, to some extent, its functional behaviors, thus housing high potential for biotechnological applications.

Over the years, mimochromes, a class of miniaturized porphyrin-based metalloproteins, have proven to be reliable but still versatile scaffolds. After two decades from their birth, we retrospectively review our work in mimochrome design and engineering, which allowed us developing functional models. They act as electron-transfer miniproteins or more elaborate artificial metalloenzymes, endowed with peroxidase, peroxygenase, and hydrogenase activities. Mimochromes represent simple yet functional synthetic models that respond to metal ion replacement and noncovalent modulation of the environment, similarly to natural heme-proteins. More recently, we have demonstrated that the most active analogue retains its functionality when immobilized on nanomaterials and surfaces, thus affording bioconjugates, useful in sensing and catalysis. This review also briefly summarizes the most important contributions to heme-protein design from leading groups in the field.

Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetrahedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assembly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. Finally, NMR data of the Zn(II)-METP3 complex, together with a retrostructural analysis of the Cys-X-X-His motif in metalloproteins, allowed us to define the model structure. All the results establish the suitability of the short METP sequence for accommodating tetrahedral metal binding sites, regardless of the first coordination ligands.

Manganese-porphyrins are important tools in catalysis, due to their capability to promote a wide variety of synthetically valuable transformations. Despite their great reactivity, the difficulties to control the reaction selectivity and to protect the catalyst from self-degradation hamper their practical application. Compared to small-molecule porphyrin complexes, metalloenzymes display remarkable features, because the reactivity of the metal center is finely modulated by a complex interplay of interactions within the protein matrix. In the effort to combine the catalytic potential of manganese porphyrins with the unique properties of biological catalysts, artificial metalloenzymes have been reported, mainly by incorporation of manganese-porphyrins into native protein scaffolds. Here we describe the spectroscopic and catalytic properties of Mn-Mimochrome VI*a (Mn-MC6*a), a mini-protein with a manganese deuteroporphyrin active site within a scaffold of two synthetic peptides covalently bound to the porphyrin. Mn-MC6*a is an efficient catalyst endowed with peroxygenase activity. The UV-vis absorption spectrum of Mn-MC6*a resembles that of Mn-reconstituted horseradish peroxidase (Mn-HRP), both in the resting and high-valent oxidized states. Remarkably, Mn-MC6*a shows a higher reactivity compared to Mn-HRP, because higher yields and chemoselectivity were observed in thioether oxidation. Experimental evidences also provided indications on the nature of the high-valent reactive intermediate and on the sulfoxidation mechanism.

Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on the recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization processes and protein redesign. Considerable progress in these diverse design approaches has produced many metal-containing biocatalysts able to adopt the functions of native enzymes or even novel functions beyond those found in Nature.

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-sulphur 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-Zn(2+) 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 Zn(2+) 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.

Rational design provides an attractive strategy to tune and control the reactivity of bioinspired catalysts. While 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. Here we show that the simple incorporation of two 2-amino isobutyric acids into an artificial peptide-based peroxidase resulted in a new catalyst (FeIII-MC6*a), with higher resistance against oxidative damage, and higher catalytic efficiency. A two-fold enhancement of the turnover number, respect to its predecessor, was observed. These results point out the protective role exerted by the peptide matrix and pave the way to the synthesis of robust bioinspired catalysts.

Enzyme-based electrodes represent an important class of biosensors that allow to electrochemically measure the products derived from a target analyte transformation. Enzymatic electrochemical biosensors, capable to detect glucose and lactate, find application in monitoring diseases, such as diabetes. This work is focused onto the development of an amperometric multy-analyte (glucose and lactate) biosensor based on glucose and lactate oxidase immobilization, loaded into photo-patternable hydrogels. The photo-definable hydrogels act as excellent biocompatible matrices providing near-physiological conditions that minimize protein denaturation. This methodology also allows biosensor miniaturization for a continuous non-invasive glucose/lactate monitoring.

Synthetic proteins represent useful tools for reproducing metalloprotein functions in minimal, well-defined scaffolds. Herein, we describe the rational refinement of function into heme-protein models from the Mimochrome family. Originally designed to mimic the bis-His cytochrome b, the Mimochrome structure was modified to introduce a peroxidase-like activity, by creating a distal cavity on the heme. The success with the first asymmetric system, Mimochrome VI (MC6), gave the opportunity to explore further modifications in order to improve the catalytic activity. Starting from ferric MC6, single amino acid substitutions were introduced in the peptide chains to obtain four compounds, which were screened for peroxidase activity. The detailed structural and functional analysis of the best analogue, Fe(III)-E(2)L(TD)-MC6, indicates that an arginine residue in proximity to the heme-distal site could assist with catalysis by favoring the formation of the intermediate "compound I", thus mimicking R(38) in HRP. This result highlights the potential of using small scaffolds for exploring the main factors that tune the heme-protein activity, and for programming new desired functions.

We set up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers (BCPs) self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we employed a mixture of polystyrene-block-poly(l-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar domains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with H2O2 as substrate. The large surface area, the tailored pore sizes, and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability.

The present invention relates to nitrogen containing macrocycles, porphyrin-like, functionalised with peptide based synthetic compounds, and their preparation and use as catalysts in fine chemistry. They can be used, for example, as catalysts in aqueous or water-alcohol solutions for peroxidation, oxidation, hydroxylation, phenol nitration and inert compound epoxidation reactions, in the control and decontamination of waters and in laboratory diagnostics. They can also be used, for example, in immunohistochemical tests, in situ hybridisation, ELISA tests, and Southern, Northern and Western blot tests. The compounds according to the invention can be used "as is" or in covalent association with biomolecules such as antibodies, antigens, enzymes, receptors, nucleic acids, peptides and proteins. The compounds according to the invention have a wide range of applications, because they represent low molecular weight (2000-5000 amu) analogues of natural and mutated haemoproteins, and are therefore proposed as particularly advantageous alternatives to products of expression or extraction. The compounds according to the invention can also be used supported on solid matrices and/or on paramagnetic and non-paramagnetic nanoparticles.

Metalloproteins utilize O-2 as an oxidant, and they often achieve a 4-electron reduction without H2O2 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 O-2 has not been reported yet. We report the construction of a diiron-binding four-helix bundle, made up of two different covalently linked (2) 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 H2O2 in the oxidation of 4-aminophenol in the presence of O-2, whereas Fe-III-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.

A cost-effective immunosensor for the detection and isolation of dental pulp stem cells (DPSCs) based on a quartz crystal microbalance (QCM) has been developed. The recognition mechanism relies on anti-CD34 antibodies, DPSC-specific monoclonal antibodies that are anchored on the surface of the quartz crystals. Due to its high specificity, real time detection, and low cost, the proposed technology has a promising potential in the field of cell biology, for the simultaneous detection and sorting of stem cells from heterogeneous cell samples. The QCM surface was properly tailored through a biotinylated self-assembled monolayer (SAM). The biotin–avidin interaction was used to immobilize the biotinylated anti-CD34 antibody on the gold-coated quartz crystal. After antibody immobilization, a cellular pellet, with a mixed cell population, was analyzed; the results indicated that the developed QCM immunosensor is highly specific, being able to detect and sort only CD34+ cells. Our study suggests that the proposed technology can detect and efficiently sort any kind of cell from samples with high complexity, being simple, selective, and providing for more convenient and time-saving operations.

Bioconjugation is a rapidly expanding field because of the numerous potential applications of bioconjugate materials. We explored the usefulness of branched porphyrins as rigid scaffolds, bearing multiple sites for bioconjugation. To this end, we first selected the tetrakis(p-(aminomethyl) phenyl) porphyrin (TAMPP) macrocycle and developed a straightforward synthetic protocol, able to provide the desired tetraphenylporphyrin, carrying four functional amino groups. The partially protection of the amino groups by tert-butoxy-carbonyl allowed the selective and specific decoration of the porphyrin with different peptide sequences. To explore the utility of the macrocycle as molecular scaffold for bioconjugation, we selected peptide sequences able to function as thrombin inhibitors. In particular, two peptide sequences, named CS3 and ES7, able to interact, respectively, with the thrombin catalytic site and the fibrinogen recognition exosite, were joined onto the porphyrin macrocycle, providing the multisite-directed inhibitor CS3-TAMPP-ES7. This multisite inhibitor and its Mn III complex are able to inhibit α-thrombin-catalyzed hydrolysis of Tos-Gly-Pro-Arg-nitroanilide with inhibition constants in the micromolar range, as well as the hydrolysis of the natural substrate fibrinogen. The inhibitor is resistant against enzymatic degradation by thrombin and is highly selective. The Mn III complex is capable of interacting with clot-bound thrombin and partially inhibits clot growth in the presence of fibrinogen. The results herein reported are very promising, suggesting the potential of the newly developed conjugate as new imaging agents for clot detection. C

Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on the recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization processes and protein redesign. Considerable progress in these diverse design approaches has produced many metal-containing biocatalysts able to adopt the functions of native enzymes or even novel functions beyond those found in Nature.

De novo design has proven a powerful methodology for understanding protein folding and function, and for mimicking or even bettering the properties of natural proteins. Extensive progress has been made in the design of helical bundles, simple structural motifs that can be nowadays designed with a high degree of precision. Among helical bundles, the four-helix bundle is widespread in nature, and is involved in numerous and fundamental processes. Representative examples are the carboxylate bridged diiron proteins, which perform a variety of different functions, ranging from reversible dioxygen binding to catalysis of dioxygen-dependent reactions, including epoxidation, desaturation, monohydroxylation, and radical formation. The “Due Ferri” (two-irons; DF) family of proteins is the result of a de novo design approach, aimed to reproduce in minimal four-helix bundle models the properties of the more complex natural diiron proteins, and to address how the amino acid sequence modulates their functions. The results so far obtained point out that asymmetric metal environments are essential to reprogram functions, and to achieve the specificity and selectivity of the natural enzymes. Here, we describe a design method that allows constructing asymmetric four-helix bundles through the covalent heterodimerization of two different α-helical harpins. In particular, starting from the homodimeric DF3 structure, we developed a protocol for covalently linking the two α 2 monomers by using the Cu(I) catalyzed azide–alkyne cycloaddition. The protocol was then generalized, in order to include the construction of several linkers, in different protein positions. Our method is fast, low cost, and in principle can be applied to any couple of peptides/proteins we desire to link.

The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV-1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure-guided approach to design peptide mimetics of the N-loop and β1-strand regions of regulated on activation normal T-cell-expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV-1 blockade. Rational modifications were sequentially introduced into the N-loop/β1-strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5-specific HIV-1 isolates (IC50 range: 104–640 nM) but lacking activity against CXCR4-specific HIV-1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the β-extended conformation adopted in full-length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in silico-optimized linker segment designed using de novo structure-prediction algorithms to stabilize the C-terminal α-helix and experimentally validated by NMR. Our mimetics exerted CCR5-antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV-1 entry inhibitors.—Lusso, P., Vangelista, L., Cimbro, R., Secchi, M., Sironi, F., Longhi, R., Faiella, M., Maglio, O., Pavone, V. Molecular engineering of RANTES peptide mimetics with potent anti-HIV-1 activity.

Heavy metals are hazardous environmental contaminants, often highly toxic even at extremely low concentrations. Monitoring their presence in environmental samples is an important but complex task that has attracted the attention of many research groups. We have previously developed a fluorescent peptidyl sensor, dH3w, for monitoring Zn2+ in living cells. This probe, designed on the base on the internal repeats of the human histidine rich glycoprotein, shows a turn on response to Zn2+ and a turn off response to Cu2+. Other heavy metals (Mn2+, Fe2+, Ni2+, Co2+, Pb2+ and Cd2+) do not interfere with the detection of Zn2+ and Cu2+. Here we report that dH3w has an affinity for Hg2+ considerably higher than that for Zn2+ or Cu2+, therefore the strong fluorescence of the Zn2+/dH3w complex is quenched when it is exposed to aqueous solutions of Hg2+, allowing the detection of sub-micromolar levels of Hg2+. Fluorescence of the Zn2+/dH3w complex is also quenched by Cu2+ whereas other heavy metals (Mn2+, Fe2+, Ni2+, Co2+, Cd2+, Pb2+, Sn2+ and Cr3+) have no effect. The high affinity and selectivity suggest that dH3w and the Zn2+/dH3w complex are suited as fluorescent sensor for the detection of Hg2+ and Cu2+ in environmental as well as biological samples. © 2018 Siepi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Editorial for this special issue of Biopolymers dedicated to Prof. Vincenzo Pavone, in celebration of his 65th birthday. The reviews and original articles included in this issue are contributed by friends, collaborators and former students of Vincenzo. They pay tribute to his outstanding career, research accomplishments, and valuable role as mentor and inspiring scientist.

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.

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.

The crystal and molecular structure of the fully protected dipeptide Boc-Val-(S)-alpha-MeSer-OMe has been determined by X-ray diffraction techniques. Crystals grown from ethyl acetate/n-pentane mixtures are tetragonal, space group I4(1), with cell parameters at 295 K of a = 15.307(2), c = 18.937(10)angstrom, V = 4437.1 angstrom3, M.W. = 332.40, Z = 8, D(m) = 0.99 g/cm3 and D(x) = 0. 995 g/cm3. The structure was solved by application of direct methods and refined to an R value of 0.028 for 1773 reflections with I greater-than-or-equal 3sigma(I) collected on a CAD-4 diffractometer. Both chiral centers have the (S) configuration. The dipeptide assumes in the solid state an S shape. The urethane moiety is in the cis conformation, while the amide bond is in the common trans conformation. The conformational angles phi1, psi1 of the Val and phi2, and psi2 of the (S)-alphaMeSer fall in the F region of the phi-psi map. The isopropyl side chain of the Val residue has the (t, g-) conformation, while the Ser side chain has a g+ conformation. The hydrogen bond donor groups are all involved in intermolecular H-bond interactions. Along the quaternary axis the dipeptide molecules are linked to each other with the formation of infinite rows.

A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technology, such as in degradation of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochemical functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biology, with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-containing proteins, and we developed models for heme proteins and diiron–oxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins.

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal-binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four-helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron-oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal-binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four-helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

An empirical equation, describing the relationship between the porphyrin methyl hyperfine shifts and the position of the axial ligand(s), has been applied to an artificial heme-protein in order to obtain insight into the active site properties of heme-protein models.

This work is based on previous evidence showing that chemotactic sequence of the urokinase receptor (uPAR88-92) drives angiogenesis in vitro and in vivo in a protease-independent manner, and that the peptide Ac-Arg-Glu-Arg-Phe-NH2 (RERF) prevents both uPAR88–92- and VEGF-induced angiogenesis. New N-acetylated and C-amidated peptide analogues containing α-methyl α-amino acids were designed and synthesized to optimize the biochemical properties for therapeutic applications. Among these, Ac-L-Arg-Aib-L-Arg-D-Cα(Me)Phe-NH2, named UPARANT, adopts in solution a turned conformation similar to that found for RERF, is stable to sterilization in 3 mg/mL sealed vials in autoclave for 20 minutes at 120°C, is stable in blood, and displays a long-time resistance to enzymatic proteolysis. UPARANT competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the formyl-peptide receptor, inhibits VEGF-directed endothelial cell migration, and prevents cytoskeletal organization and αvβ3 activation in endothelial cells exposed to VEGF. In vitro, UPARANT inhibits VEGF-dependent tube formation of endothelial cells at a 100× lower concentration than RERF. In vivo, UPARANT reduces to the basal level VEGF-dependent capillary sprouts originating from the host vessels that invaded Matrigel sponges implanted in mice, and completely prevents neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Both excellent stability and potency position UPARANT as a promising new therapeutic agent for the control of diseases fueled by excessive angiogenesis, such as cancer and inflammation. Mol Cancer Ther; 13(5); 1092–104. ©2014 AACR.

The crystal structures of three fully protected tripeptides containing the Dϕg residue (Cα,α-diphenylglycine) in the central position are reported, namely Z-Gly-Dϕg-Gly-OMe (a), Z-Gly-Dϕg-Aib-OMe (b) and Z-Aib-Dϕg-Aib-OMe (c). The molecular conformations are quite unusual because the Dϕg residue adopts a folded conformation in the 310-helical region when the following residue adopts a folded conformation of opposite handedness (peptidesbandc). In contrast, the Dϕg residue adopts the more frequently observed fully extended conformation when the following residue adopts a semi-extended conformation (peptidea). These findings are in agreement with the theoretical calculations on Ac-Dϕg-Aib-NHCH3 and Ac-Aib-Dϕg-NHCH3 also reported in this work. © 1998 European Peptide Society and John Wiley & Sons, Ltd.

In the present paper we describe the solution nmr structural analysis and restrained molecular dynamic simulation of the cyclic pentapeptide cyclo-(Pro-Phe-Phe-beta-Ala-beta-Ala). The conformational analysis carried out in CD3CN and dimethylsulfoxide (DMSO) solutions by nmr spectroscopy was based on interproton distances derived from rotating frame nuclear Overhauser effect spectroscopy spectra and homonuclear coupling constants. A restrained molecular dynamic simulation in vacuo was also performed to build refined molecular models. The molecule is present in both solvent systems as two slowly interconverting conformers, characterized by a cis-trans isomerism around the beta-Ala5-Pro1 peptide bond. In CD3CN solution, the conformer with a ci5 peptide bond is quite similar to that observed in the solid state, while the conformer containing all trans peptide bonds is characterized by an intramolecular hydrogen bond stabilizing a C10- and a C13-ring structure. In DMSO solution, the trans isomer is partly similar to that observed in CD3CN solution while the cis isomer is different from that observed in the solid state. The effect of the solvent in stabilizing different conformations was also investigated in DMSO-CD3CN solvent mixtures.

We recently reported the rational design, synthetics, and structural characterization of the most potent and selective peptide-based neurokinin A antagonist thus far described: cyclo(Met1-Asp2-Trp3-Phe4-Dap5-Leu6)cyclo(2 beta-5 beta). Its bicyclic structure is characterized by a type I and a type II two beta-turn around Trp3-Phe4 and Leu6-Met1, respectively. In order to understand whether the two different beta-turned structures are determined by the bicyclic structure or by the amino acid type at the corner positions, we have synthesized the pseudo-symmetrical analogue cyclo(Phe1-Asp2-Trp3-Phe4-Dap5-Trp6)cyclo(2 beta-5 beta). The structural characterization in the crystal state and in solution, here reported, gives an experimental evidence that the backbone of the bicyclic structure is a rigid scaffold that can be used to build both a type I and type II beta-turn independently from the amino acid composition.