ABSTRACT
In the field of nucleic acid therapy there is major interest in the development of libraries of DNA-reactive small molecules which are tethered to vectors that recognize and bind specific genes. This approach mimics enzymatic gene editors, such as ZFNs, TALENs and CRISPR-Cas, but overcomes the limitations imposed by the delivery of a large protein endonuclease which is required for DNA cleavage. Here, we introduce a chemistry-based DNA-cleavage system comprising an artificial metallo-nuclease (AMN) that oxidatively cuts DNA, and a triplex-forming oligonucleotide (TFO) that sequence-specifically recognises duplex DNA. The AMN-TFO hybrids coordinate CuII ions to form chimeric catalytic complexes that are programmable - based on the TFO sequence employed - to bind and cut specific DNA sequences. Use of the alkyne-azide cycloaddition click reaction allows scalable and high-throughput generation of hybrid libraries that can be tuned for specific reactivity and gene-of-interest knockout. As a first approach, we demonstrate targeted cleavage of purine-rich sequences, optimisation of the hybrid system to enhance stability, and discrimination between target and off-target sequences. Our results highlight the potential of this approach where the cutting unit, which mimics the endonuclease cleavage machinery, is directly bound to a TFO guide by click chemistry.
Subject(s)
Copper/metabolism , DNA/metabolism , Endonucleases/metabolism , Metalloproteins/metabolism , Oligonucleotides/metabolism , Click Chemistry , Copper/chemistry , DNA/chemistry , Metalloproteins/chemical synthesis , Metalloproteins/chemistry , Molecular Structure , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistryABSTRACT
To mimic a hypothetical pathway for protein evolution, we previously tailored a monomeric protein (cyt cb562 ) for metal-mediated self-assembly, followed by re-design of the resulting oligomers for enhanced stability and metal-based functions. We show that a single hydrophobic mutation on the cyt cb562 surface drastically alters the outcome of metal-directed oligomerization to yield a new trimeric architecture, (TriCyt1)3. This nascent trimer was redesigned into second and third-generation variants (TriCyt2)3 and (TriCyt3)3 with increased structural stability and preorganization for metal coordination. The three TriCyt variants combined furnish a unique platform to 1)â provide tunable coupling between protein quaternary structure and metal coordination, 2)â enable the construction of metal/pH-switchable protein oligomerization motifs, and 3)â generate a robust metal coordination site that can coordinate all mid-to-late first-row transition-metal ions with high affinity.
Subject(s)
Metalloproteins/chemical synthesis , Metals, Heavy/chemistry , Hydrophobic and Hydrophilic Interactions , Metalloproteins/chemistry , Models, MolecularABSTRACT
We have examined the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resistance regulator (LmrR) by stop codon suppression methodology. LmrR_HQAla was shown to complex efficiently with three different metal ions, CuII , ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII -bound LmrR_HQAla enzyme was shown through its ability to catalyse asymmetric Friedel-Craft alkylation and water addition, whereas the ZnII -coupled enzyme was shown to mimic natural Zn hydrolase activity.
Subject(s)
Alanine/chemistry , Drug Design , Hydroxyquinolines/chemistry , Metalloproteins/chemical synthesis , Metals, Heavy/chemistry , Alanine/analogs & derivatives , Catalysis , Metalloproteins/chemistry , Models, Molecular , Molecular StructureABSTRACT
Enzyme biosensors are useful tools that can monitor rapid changes in metabolite levels in real-time. However, current approaches are largely constrained to metabolites within a limited chemical space. With the rising development of artificial metalloenzymes (ArM), a unique opportunity exists to design biosensors from the ground-up for metabolites that are difficult to detect using current technologies. Here we present the design and development of the ArM ethylene probe (AEP), where an albumin scaffold is used to solubilize and protect a quenched ruthenium catalyst. In the presence of the phytohormone ethylene, cross metathesis can occur to produce fluorescence. The probe can be used to detect both exogenous- and endogenous-induced changes to ethylene biosynthesis in fruits and leaves. Overall, this work represents an example of an ArM biosensor, designed specifically for the spatial and temporal detection of a biological metabolite previously not accessible using enzyme biosensors.
Subject(s)
Biomimetic Materials/chemical synthesis , Biosensing Techniques/instrumentation , Ethylenes/analysis , Metalloproteins/metabolism , Plant Growth Regulators/analysis , Actinidia/metabolism , Arabidopsis/metabolism , Catalysis , Chemistry Techniques, Synthetic/methods , Enzymes/chemical synthesis , Enzymes/metabolism , Ethylenes/metabolism , Fluorescence , Fruit/metabolism , Gases/analysis , Gases/metabolism , Metalloproteins/chemical synthesis , Plant Growth Regulators/metabolism , Plant Leaves/chemistry , Ruthenium/chemistry , Serum Albumin, Human/chemical synthesis , Serum Albumin, Human/metabolismABSTRACT
This review describes the recent advances in the design of novel artificial metalloenzymes and their application in C-H activation reactions. The combination of enzymes and metal or organometallic complexes for the creation of new artificial metalloenzymes has represented a very exciting research line. In particular, the development of proteins with the ability to perform C-H functionalization presents a significant challenge. Here we discuss the development of these processes on natural metalloenzymes by using directed evolution, biotin-(strept)avidin technologies, photocatalytic hybrids or reconstitution of heme-protein technology.
Subject(s)
Metalloproteins/antagonists & inhibitors , Organometallic Compounds/pharmacology , Metalloproteins/chemical synthesis , Metalloproteins/chemistry , Models, Molecular , Molecular Structure , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistryABSTRACT
Metal-binding peptides are versatile building blocks in supramolecular chemistry. We recently reported a class of crystalline materials formed through a combination of coiled-coil peptide self-association and metal coordination. Here, we probe the serendipitously discovered metal binding motif that drives the assembly and apply these insights to exert rational control over structure and morphology in the materials.
Subject(s)
Metalloproteins/chemistry , Amino Acid Motifs , Amino Acid Sequence , Copper/chemistry , Electron Spin Resonance Spectroscopy/methods , Metalloproteins/chemical synthesis , Protein Engineering/methods , Protein Multimerization , Pyridines/chemistryABSTRACT
We report the experimental results of a study of the electron-transfer processes of redox-active metalloproteins bound to mixed self-assembled monolayers (SAMs) on magnetic (nickel or ultrathin gold-coated nickel) or nonmagnetic (gold) electrodes. Metalloproteins, such as hemoglobin (Hb), Cytochromeâ C (Cytâ C), and Cytâ C oxidase, are attached through electrostatic interactions to the free carboxylate or imidazole groups present in the mixed SAMs. The formation of both mixed SAMs and SAM/metalloprotein heterostructures were confirmed by using advanced surface analysis techniques, such as polarization modulation infrared reflection absorption spectroscopy and aqueous contact angle measurements. Electrochemical measurements indicated a stronger electronic coupling between Hb and Cytâ C oxidase and the mixed-SAM-coated gold or gold-coated-nickel electrodes, whereas a weaker coupling was found between the protein and the pure nickel electrode. Surface coverage and the electron-transfer rate constant were estimated from the cyclic voltammetry data.
Subject(s)
Electrochemical Techniques , Ferric Compounds/chemistry , Metalloproteins/chemistry , Electrodes , Gold/chemistry , Metalloproteins/chemical synthesisABSTRACT
A cytochrome P450 was engineered to selectively incorporate Ir(Me)-deuteroporphyrinâ IX (Ir(Me)-DPIX), in lieu of heme, in bacterial cells. Cofactor selectivity was altered by introducing mutations within the heme-binding pocket to discriminate the deuteroporphyrin macrocycle, in combination with mutations to the P450 axial cysteine to accommodate a pendant methyl group on the Ir(Me) center. This artificial metalloenzyme was investigated for activity in non-native metallocarbenoid-mediated olefin cyclopropanation reactions and showed enhanced activity for aliphatic and electron-deficient olefins when compared to the native heme enzyme. This work provides a general strategy to augment the chemical functionality of heme enzymes in cells with application towards abiotic catalysis.
Subject(s)
Biocatalysis , Metalloproteins/chemical synthesis , Protein Engineering , Bacteria/cytology , Cytochrome P-450 Enzyme System/chemistry , Deuterium , Heme/chemistry , Iridium/chemistry , Porphyrins/chemistryABSTRACT
To better understand metalloproteins with Mn-clusters, we have designed artificial four-helix bundles to have one, two, or three dinuclear metal centers able to bind Mn(II). Circular dichroism measurements showed that the Mn-proteins have substantial α-helix content, and analysis of electron paramagnetic resonance spectra is consistent with the designed number of bound Mn-clusters. The Mn-proteins were shown to catalyze the conversion of hydrogen peroxide into molecular oxygen. The loss of hydrogen peroxide was dependent upon the concentration of protein with bound Mn, with the proteins containing multiple Mn-clusters showing greater activity. Using an oxygen sensor, the oxygen concentration was found to increase with a rate up to 0.4µM/min, which was dependent upon the concentrations of hydrogen peroxide and the Mn-protein. In addition, the Mn-proteins were shown to serve as electron donors to bacterial reaction centers using optical spectroscopy. Similar binding of the Mn-proteins to reaction centers was observed with an average dissociation constant of 2.3µM. The Mn-proteins with three metal centers were more effective at this electron transfer reaction than the Mn-proteins with one or two metal centers. Thus, multiple Mn-clusters can be incorporated into four-helix bundles with the capability of performing catalysis and electron transfer to a natural protein.
Subject(s)
Manganese/chemistry , Metalloproteins/chemistry , Oxygen/chemistry , Protein Conformation, alpha-Helical , Binding Sites , Circular Dichroism , Electron Spin Resonance Spectroscopy , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Metalloproteins/chemical synthesis , Metalloproteins/metabolism , Models, Molecular , Protein BindingABSTRACT
An efficient synthesis of the molybdopterin/molybdenum cofactor (Moco) oxidation product dephospho Formâ A is described that assembles the pteridinone system starting from an iodinated aminopyrazine. The sodium salt of dephospho Formâ A could be purified by precipitation from methanol, which paved the way to the title compound in the 100â mg range. By HPLC, the synthetic material was compared with a sample isolated from a recombinant Moco containing protein. Analysis of dephospho Formâ A is the only method that allows the quantification of the Moco content of crude cell extracts and recombinant protein preparations.
Subject(s)
Coenzymes/chemistry , Metalloproteins/chemistry , Pteridines/chemistry , Pyrazines/chemistry , Coenzymes/chemical synthesis , Fungal Proteins/metabolism , Metalloproteins/chemical synthesis , Molybdenum Cofactors , Neurospora crassa/enzymology , Nitrate Reductase/metabolism , Oxidation-Reduction , Pteridines/chemical synthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purificationABSTRACT
The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a d-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CS sequence, yielding a GRAND-CoilSer (GRAND-CS) to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination, and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled-coil designs.
Subject(s)
Amino Acids/chemistry , Metalloproteins/chemistry , Peptides/chemistry , Protein Structure, Secondary , Amino Acid Sequence/genetics , Amino Acids/genetics , Binding Sites , Crystallography, X-Ray , Ligands , Metalloproteins/chemical synthesis , Metalloproteins/genetics , Metals , Models, MolecularABSTRACT
Complete thermodynamic descriptions of the interactions of cofactors with proteins via equilibrium studies are challenging, but are essential to the evaluation of designed metalloproteins. While decades of studies on protein-protein interaction thermodynamics provide a strong underpinning to the successful computational design of novel protein folds and de novo proteins with enzymatic activity, the corresponding paucity of data on metal-protein interaction thermodynamics limits the success of computational metalloprotein design efforts. By evaluating the thermodynamics of metal-protein interactions via equilibrium binding studies, protein unfolding free energy determinations, proton competition equilibria, and electrochemistry, a more robust basis for the computational design of metalloproteins may be provided. Our laboratory has shown that such studies provide detailed insight into the assembly and stability of designed metalloproteins, allow for parsing apart the free energy contributions of metal-ligand interactions from those of porphyrin-protein interactions in hemeproteins, and even reveal their mechanisms of proton-coupled electron transfer. Here, we highlight studies that reveal the complex interplay between the various equilibria that underlie metalloprotein assembly and stability and the utility of making these detailed measurements.
Subject(s)
Hemeproteins/chemistry , Metalloproteins/chemistry , Metals/chemistry , Protein Engineering/methods , Binding Sites , Computational Biology/methods , Energy Metabolism , Ligands , Metalloproteins/chemical synthesis , Protein Unfolding , Protons , ThermodynamicsABSTRACT
Artificial metalloenzymes represent an attractive means of combining state-of-the-art transition metal catalysis with the benefits of natural enzymes. Despite the tremendous recent progress in this field, current efforts toward the directed evolution of these hybrid biocatalysts mainly rely on the laborious, individual purification of protein variants rendering the throughput, and hence the outcome of these campaigns feeble. We have recently developed a screening platform for the directed evolution of artificial metalloenzymes based on the streptavidin-biotin technology in the periplasm of the Gram-negative bacterium Escherichia coli. This periplasmic compartmentalization strategy comprises a number of compelling advantages, in particular with respect to artificial metalloenzymes, which lead to a drastic increase in the throughput of screening campaigns and additionally are of unique value for future in vivo applications. Therefore, we highlight here the benefits of this strategy and intend to propose a generalized guideline for the development of novel transition metal-based biocatalysts by directed evolution in order to extend the natural enzymatic repertoire.
Subject(s)
Directed Molecular Evolution , Enzymes/chemistry , Metalloproteins/chemistry , Periplasm/chemistry , Catalysis , Enzymes/chemical synthesis , Enzymes/genetics , Metalloproteins/chemical synthesis , Metalloproteins/genetics , Metals/chemistry , Periplasm/genetics , Protein EngineeringABSTRACT
Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme-nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedures on how to choose the protein scaffold, design a heterobinuclear metal center in the protein scaffold computationally, incorporate metal ions into the protein, and characterize the resulting metalloproteins, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity.
Subject(s)
Heme/chemistry , Metalloproteins/chemistry , Myoglobin/chemistry , Protein Engineering/methods , Catalysis , Cytochrome-c Peroxidase/chemistry , Heme/chemical synthesis , Ions/chemistry , Metalloproteins/chemical synthesis , Metals/chemistry , Oxidation-ReductionABSTRACT
A solid-phase strategy using lipase as a biomolecular scaffold to produce a large amount of Cu(2+)-metalloenzyme is proposed here. The application of this protocol on different 3D cavities of the enzyme allows creating a heterogeneous artificial metallolipase showing chimeric catalytic activity. The artificial catalyst was assessed in Diels-Alder cycloaddition reactions and cascade reactions showing excellent catalytic properties.
Subject(s)
Lipase/chemical synthesis , Metalloproteins/chemical synthesis , Catalysis , Catalytic Domain , Lipase/chemistry , Metalloproteins/chemistry , Models, MolecularABSTRACT
The ability of many copper metalloenzymes to activate O2 and transfer it to organic substrates has motivated extensive attention in the literature. Investigations focusing on synthetic analogues have provided a detailed understanding of the structures of potential intermediates, thereby helping to guide mechanistic studies. We report herein a crystallographically characterized synthetic Cu(II)2(µ-η(1):η(1)-O2) complex exhibiting cis-peroxo bonding geometry, known in iron chemistry but previously unobserved for copper. Detailed investigation by UV-vis, resonance Raman, and infrared spectroscopies provides evidence for a significantly diminished copper-oxygen interaction (ε ≈ 3000 M(-1) cm(-1), ν(Cu-O) = 437 cm(-1), ν(O-O) = 799 cm(-1)) relative to those in known 'coupled' Cu2O2 species, consistent with magnetic measurements which show that the peroxide mediates only weak antiferromagnetic coupling (-2J = 144 cm(-1)). These characteristics are comparable with those of a computationally predicted transition state for O2 binding to type 3 copper centers, providing experimental evidence for the proposed mechanism of O2 activation and supporting the biological relevance of the Cu(II)2(µ-η(1):η(1)-O2) cis-species. The peroxide bonding arrangement also allows binding of sodium cations, observed both in the solid state and in solution. Binding induces changes on an electronic level, as monitored by UV-vis spectroscopy (K(a) = 1700 M(-1)), reminiscent of redox-inactive metal binding by iron-oxygen species. The results presented highlight the analogous chemistry these reactive oxygen species undergo, with respect to both their mechanism of formation, and the molecular interactions in which they participate.
Subject(s)
Copper/metabolism , Metalloproteins/metabolism , Oxygen/metabolism , Binding Sites , Copper/chemistry , Crystallography, X-Ray , Ions/chemistry , Ions/metabolism , Metalloproteins/chemical synthesis , Metalloproteins/chemistry , Models, Molecular , Molecular Conformation , Oxygen/chemistryABSTRACT
A new metalloenzyme formed by a Fe(III)-mesoporphyrin IX functionalized by two helical decapeptides was synthesized to mimic function and structural features of a hemoprotein active site. Each decapeptide comprises six 2-aminoisobutyric acid residues, which constrain the peptide to attain a helical conformation, and three glutamic residues for improving the solubility of the catalyst in aqueous solutions. The new compound shows a marked amphiphilic character, featuring a polar outer surface and a hydrophobic inner cavity that hosts the reactants in a restrained environment where catalysis may occur. The catalytic activity of this synthetic mini-protein was tested with respect to the oxidation of L- and D-Dopa by hydrogen peroxide, showing moderate stereoselectivity. Structural information on the new catalyst and its adduct with the L- or D-Dopa substrate were obtained by the combined use of spectroscopic techniques and molecular mechanics calculations.
Subject(s)
Hemeproteins/chemical synthesis , Mesoporphyrins/chemistry , Metalloproteins/chemical synthesis , Peroxidases/chemical synthesis , Catalytic Domain , Dihydroxyphenylalanine/chemistry , Kinetics , Models, Molecular , Molecular Mimicry , Oligopeptides/chemistry , Oxidation-Reduction , Spectroscopy, Fourier Transform Infrared , Stereoisomerism , ThermodynamicsABSTRACT
The synthesis and stereochemical assignment of two classes of iron-containing nucleoside analogues, both of which contain a butadiene-Fe(CO)3 substructure, is described. The first type of compounds are Fe(CO)3-complexed 3'-alkenyl-2',3'-dideoxy-2',3'-dehydro nucleosides (2,5-dihydrofuran derivatives), from which the second class of compounds is derived by formal replacement of the ring oxygen atom by a CH2 group (carbocyclic nucleoside analogues). These compounds were prepared in a stereoselective manner through the metal-assisted introduction of the nucleobase. Whilst the furanoid intermediates were prepared from carbohydrates (such as methyl-glucopyranoside), the carbocyclic compounds were obtained by using an intramolecular Pauson-Khand reaction. Stereochemical assignments based on NMR and CD spectroscopy were confirmed by X-ray structural analysis. Biological investigations revealed that several of the complexes exhibited pronounced apoptosis-inducing properties (through an unusual caspase 3-independent but ROS-dependent pathway). Furthermore, some structure-activity relationships were identified, also as a precondition for the design and synthesis of fluorescent and biotin-labeled conjugates.
Subject(s)
Biotin/chemical synthesis , Fluorescent Dyes/chemical synthesis , Iron/chemistry , Metalloproteins/chemical synthesis , Metalloproteins/pharmacology , Nucleosides/chemical synthesis , Nucleosides/pharmacology , Apoptosis/drug effects , Biotin/chemistry , Fluorescent Dyes/chemistry , Magnetic Resonance Spectroscopy , Metalloproteins/chemistry , Molecular Structure , Nucleosides/chemistry , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2'-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein-based metal ligands, and two metal-bound water molecules. Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability to bind divalent cations including Co(2+), Zn(2+), Fe(2+), and Ni(2+), with a Kd for Zn(2+) of â¼40 pM. X-ray crystal structures of the designed protein bound to Co(2+) and Ni(2+) have RMSDs to the design model of 0.9 and 1.0 Å respectively over all atoms in the binding site.
Subject(s)
Amino Acids/chemistry , Cobalt/chemistry , Computational Biology , Metalloproteins/chemical synthesis , Metalloproteins/chemistry , Metalloproteins/isolation & purification , Models, Molecular , Molecular StructureABSTRACT
A new artificial metalloenzyme, MP3 (MiniPeroxidaseâ 3), designed by combining the excellent structural properties of four-helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix-loop-helix/heme/helix-loop-helix sandwich arrangement, characterised by 1)â a His residue on one chain that acts as an axial ligand to the iron ion; 2)â a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3)â an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP-like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix-forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe(III)-MP3 possesses peroxidase-like activity comparable to R38A-hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic.