Using Nonnatural Amino Acids to Control Metal-Coordination Number in Three-Stranded Coiled Coils (original) (raw)
Related papers
Two-Metal Ion, Ni(II) and Cu(II), Binding α-Helical Coiled Coil Peptide
Journal of the American Chemical Society, 2004
Metalloproteins are an attractive target for de novo design. Usually, natural proteins incorporate two or more (hetero-or homo-) metal ions into their frameworks to perform their functions, but the design of multiple metal-binding sites is usually difficult to achieve. Here, we undertook the de novo engineering of heterometal-binding sites, Ni(II) and Cu(II), into a designed coiled coil structure based on an isoleucine zipper (IZ) peptide. Previously, we described two peptides, IZ-3adH and IZ-3aH. The former has two His residues and forms a triple-stranded coiled coil after binding Ni(II), Zn(II), or Cu(II). The latter has one His residue, which allowed binding with Cu(II) and Zn(II), but not with Ni(II). On the basis of these properties, we newly designed IZ(5)-2a3adH as a heterometal-binding peptide. This peptide can bind Cu(II) and Ni(II) simultaneously in the hydrophobic core of the triple-stranded coiled coil. The first metal ion binding induced the folding of the peptide into the triple-stranded coiled coil, thereby promoting the second metal ion binding. This is the first example of a peptide that can bind two different metal ions. This construction should provide valuable insights for the de novo design of metalloproteins.
Design of a Three-Helix Bundle Capable of Binding Heavy Metals in a Triscysteine Environment
Angewandte Chemie International Edition, 2011
An important objective of de novo protein design is the preparation of metalloproteins, as many natural systems contain metals which play crucial roles for the function and/or structural integrity of the biopolymer.[1,2] Metalloproteins catalyze some of the most important processes in nature, ranging from energy generation and transduction to complex chemical transformations. At the same time, metals in excess can be deleterious to cells and some ions are purely toxic, having no known beneficial effects (e.g., Hg II or Pb II ). One hopes to use a first principles approach for realizing both known metallocenters and also to prepare novel sites which may lead to exciting new catalytic transformations. However, designing novel metalloproteins is a challenging and complex task, especially if one desires to prepare asymmetric metal environments.
Journal of Inorganic Biochemistry, 2013
This paper describes the design, characterization, and metal-binding properties of a 32-residue polypeptide called AQ-C16C19. The sequence of this peptide is composed of four repeats of the seven residue sequence Ile-Ala-Ala-Leu-Glu-Gln-Lys but with a Cys-X-X-Cys metal-binding motif substituted at positions 16-19. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spectroscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded α-helical coiled coil is dominant between pH 5.4 and 8.5. The Cd 2+-binding properties of the AQ-C16C19 peptide were studied by ultraviolet-visible spectroscopy (UV-vis), electrospray ionization mass spectrometry (ESI MS), and 113 Cd NMR techniques. The holoprotein was found to contain a polynuclear cadmiumthiolate center formed within the hydrophobic core of the triple-stranded α-helical coiled-coil structure. The X-ray crystal structure of the Cd-loaded peptide, resolved at 1.85 Å resolution, revealed an adamantane-like configuration of the polynuclear metal center consisting of four cadmium ions, six thiolate sulfur ligands from cysteine residues and four oxygen-donor ligands. Three of these are from glutamic acid residues and one is from an exogenous water molecule. Thus, each cadmium ion is coordinated in a distorted tetrahedral S 3 O geometry. The metal cluster was found to form cooperatively at pH 5.4 but in a stepwise fashion at pH>7. The results demonstrate that synthetic coiled-coils can be designed to incorporate multinuclear metal clusters, a proof-of-concept for their potential use in developing synthetic metalloenzymes and multi-electron redox agents.
Proceedings of the National Academy of Sciences, 2012
One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H) 3
Sculpting Metal-binding Environments in De Novo Designed Three-helix Bundles
De novo protein design is a biologically relevant approach used to study the active centers of native metalloproteins. In this review, we will first discuss the design process in achieving a 3 D, a de novo designed three-helix bundle peptide with a well-defined fold. We will then cover our recent work in functionalizing the a 3 D framework by incorporating a tris(cysteine) and tris(histidine) motif. Our first design contains the thiol-rich sites found in metalloregulatory proteins that control the levels of toxic metal ions (Hg, Cd, and Pb). The latter design recapitulates the catalytic site and activity of a natural metalloenzyme carbonic anhydrase. The review will conclude with future design goals aimed at introducing an asymmetric metal-binding site in the a 3 D framework.
Manipulation of protein-complex function by using an engineered heterotrimeric coiled-coil switch
Organic & Biomolecular Chemistry, 2009
Design methodology of variant proteins, in which original functions can be manipulated by additive ligand binding, is an attractive target of protein engineering. Especially for multi-protein complexes, techniques for constructing variants which allow the switching on or off of original functions by ligands have been limited until now. We examined a method of utilizing a de novo designed protein module, IZ-DS, which has a tertiary structure that can be significantly changed from a random coil to a folded coiled-coil structure following binding with peptide ligand, IZ-3K. By introducing a metamorphosis IZ-DS sequence to one of the components in a target multi-protein complex, the IZ-3K binding and the subsequent structural transition of the IZ-DS moiety would affect the tertiary structure of the introduced protein unit, and the function of the total multi-protein complex may also be altered. In this research, we used the T7 RNA polymerase (T7 RNAP)/T7 lysozyme complex as the target multi-protein complex, in which allosteric binding of the T7 lysozyme to T7 RNAP halts the RNA synthesis of T7 RNAP. The IZ-DS sequence was introduced to the T7 lysozyme. By optimizing the introduction site of the IZ-DS sequence in the T7 lysozyme, we succeeded in constructing the T7 lysozyme variant, DS-Lys 23. In the absence of IZ-3K, the mixture of T7 RNAP and DS-Lys 23 exhibited RNA synthesis due to the weakening of the interaction between T7 RNAP and DS-Lys 23. Whereas, after the addition of IZ-3K, RNA synthesis was significantly suppressed by the binding of DS-Lys 23 /IZ-3K complex. The present methodology using a designed ligand-dependent metamorphosis protein sequence constitutes another possible method for the de novo manipulation of various functions of natural protein complexes.
Journal of the American Chemical Society, 2007
Ribosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from adjacent noncovalently-associated helical modules. In our designs peptide self-assembly positions a cysteine residue that is used to covalently capture substrates from solution via transthiolesterification (substrate loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a covalently tethered amino acid or modeled by the ε-amine of an active site lysine. However, through systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to ~10 3-fold) by engineering changes in the active site microenvironment through amino acid substitutions and variations in the inter-residue distances and geometry. Mechanistic studies based on 15 N-NMR and kinetic analysis further indicate that certain active site constellations furnish an unexpectedly large pK a depression (1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes.
Protein Science, 2000
We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded a-helix structure. The addition of Cd~II!, however, resulted in the reconstitution of the triple-stranded a-helix bundle, as revealed by circular dichroism~CD! spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant K d was determined to be 1.5 6 0.8 mM for Cd~II!. The triple-stranded complex formed by the 113 Cd~II! ion showed a single 113 Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl Ϫ ions. The 113 Cd NMR resonance was connected with the bH protons of the cysteine residue by 1 H-113 Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg~II! also induced the assembly of the peptide into a triple-stranded a-helical bundle below the Hg~II!0peptide ratio of 103. With excess Hg~II!, however, the a-helicity of the peptide was decreased, with the change of the Hg~II! coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.
From Unnatural Amino Acid Incorporation to Artificial Metalloenzymes
2016
Studies and development of artificial metalloenzymes have developed into vibrant areas of research. It is expected that artificial metalloenzymes will be able to combine the best of enzymatic and homogenous catalysis, that is, a broad catalytic scope, high selectivity and activity under mild, aqueous conditions. Artificial metalloenzyme consist of a host protein and a newly introduced artificial metal center. The host protein merely functions as ligand controlling selectivity and augmenting reactivity, while the metal center determines the reactivity. Potential applications range from catalytic production of fine chemicals and feedstock to electron transfer utilization (e.g. fuel cells, water splitting) and medical research (e.g. metabolic screening). Particularly modern asymmetric synthesis is expected to benefit from a successful combination of the power of biocatalysis (substrate conversion via multi-step or cascade reactions, potentially immortal catalyst, unparalleled selectivity and optimization by evolutionary methods) with the versatility and mechanism based optimization methods of homogeneous catalysis. However, so far systems are either limited in structural diversity (biotin-avidin technology) or fail to deliver the selectivities expected (covalent approaches). This thesis explores a novel strategy based on the site-selective incorporation of unnatural, metal binding amino acids into a host protein. The unnatural amino acids can either serve directly as metal binding centers can be used as anchoring points for artificial metallo-cofactors. The identification expression, purification and modification of a suitable protein scaffolds