Metal-binding properties and structural characterization of a self-assembled coiled coil: Formation of a polynuclear Cd–thiolate cluster (original) (raw)
Related papers
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.
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.
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.
Journal of the American Chemical Society, 2002
Designed R-helical peptides of the TRI family with a general sequence Ac-G(LKALEEK)4G-CONH2 were used as model systems for the study of metal-protein interactions. Variants containing cysteine residues in positions 12 (TRI L12C) and 16 (TRI L16C) were used for the metal binding studies. Cd(II) binding was investigated, and the results were compared with previous and current work on Hg(II) and As(III) binding. The metal peptide assemblies were studied with the use of UV, CD, EXAFS, 113 Cd NMR, and 111m Cd perturbed angular correlation spectroscopy. The metalated peptide aggregates exhibited pHdependent behavior. At high pH values, Cd(II) was bound to the three sulfurs of the three-stranded R-helical coiled coils. A mixture of two species was observed, including Cd(II) in a trigonal planar geometry. The complexes have UV bands at 231 nm (20 600 M -1 cm -1 ) for TRI L12C and 232 nm (22 600 M -1 cm -1 ) for TRI L16C, an average Cd-S bond length of 2.49 Å for both cases, and a 113 Cd NMR chemical shift at 619 ppm (Cd II (TRI L12C)3 -) or 625 ppm (Cd II (TRI-L16C)3 -). Nuclear quadrupole interactions show that two different Cd species are present for both peptides. One species with ω0 ) 0.45 rad/ns and low η is attributed to a trigonal planar Cd-(Cys)3 site. The other, with a smaller ω0, is attributed to a four-coordinate Cd-(Cys)3(H2O) species. At low pH, no metal binding was observed. Hg(II) binding to TRI L12C was also found to be pH dependent, and a 3:1 sulfur-to-mercury(II) species was observed at pH 9.4. These metal peptide complexes provide insight into heavy metal binding and metalloregulatory proteins such as MerR or CadC.
Journal of Molecular Biology, 1998
To de®ne the delicate interplay between metal chelation, protein folding and function in metalloproteins, a family of de novo-designed peptides was synthesized that self-assemble in aqueous solution to form two and three-stranded a-helical coiled coils. Each peptide contains a single Cys residue at an a or d position of the heptad repeat. Peptide association thus produces a Cys-rich coordination environment that has been used to bind Hg(II) ions. These peptides display a pH-dependent association, with trimers observed above the pK a of Glu side-chains and dimers below this value. Finite-difference Poisson-Boltzmann calculations suggest that the dimeric state decreases the unfavorable electrostatic interactions between positively charged Lys side-chains (relative to the trimer). The Cys-containing peptides bind Hg(II) in a position-dependent fashion. Cys at a positions form three-coordinate Hg complexes at high pH where the trimeric aggregation state predominates, and two-coordinate complexes at lower pH. A d position Cys, however, is only able to generate the two-coordinate complex, illustrating the difference in coordination geometry between the two positions in the coiled coil. The binding of Hg(II) was also shown to substantially increase the stability of the helical aggregates.
Using Nonnatural Amino Acids to Control Metal-Coordination Number in Three-Stranded Coiled Coils
Angewandte Chemie International Edition, 2006
Controlling the coordination geometry of a metal center is the first step in preparing proteins capable of catalysis. Substitution of a single amino acid can shift an equilibrium of 3-and 4-coordinate Cd IIpeptide complexes to proteins containing exclusively 3-or 4-coordinate metal sites. For details, see the communication by V. L. Pecoraro and co-workers on the following pages.
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.
Cu(I) binding properties of a designed metalloprotein
Journal of Inorganic Biochemistry, 2010
The Cu(I) binding properties of the designed peptide C16C19-GGY are reported. This peptide was designed to form an a-helical coiled-coil but modified to incorporate a Cys-X-X-Cys metal-binding motif along its hydrophobic face. Absorption, emission, electrospray ionization mass spectrometry (ESI-MS), and circular dichroism (CD) experiments show that a 1:1 Cu-peptide complex is formed when Cu(I) is initially added to a solution of the monomeric peptide. This is consistent with our earlier study in which the emissive 1:1 complex was shown to exist as a peptide tetramer containing a tetranuclear copper cluster Kharenko et al. (2005) [11]. The presence of the tetranuclear copper center is now confirmed by ESI-MS which along with UV data show that this cluster is formed in a cooperative manner. However, spectroscopic titrations show that continued addition of Cu(I) results in the occupation of a second, lower affinity metal-binding site in the metallopeptide. This occupancy does not significantly affect the conformation of the metallopeptide but does result in a quenching of the 600 nm emission. It was further found that the exogenous reductant tris(2-carboxyethyl)phosphine (TCEP) can competitively inhibit the binding of Cu(I) to the low affinity site of the peptide, but does not interact with Cu(I) clusters.
Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex
Chemistry (Weinheim an der Bergstrasse, Germany), 2017
The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was...
Inorganic Chemistry, 2008
A two-stranded R-helical coiled coil was prepared having a Cys 4 metal-binding site within its hydrophobic interior. The addition of Cd 2+ results in the incorporation of 2 equiv of metal ion, which is accompanied by a conformational change of the peptide, as observed by circular dichroism (CD) spectroscopy. Isothermal titration calorimetry (ITC) shows that the addition of Cd 2+ is accompanied by two thermodynamic events. A comparison of the time dependence of the ITC behavior with those of the UV absorption and CD behavior allows the assignment of these events to a preliminary endothermic metal-binding step followed by a slower exothermic conformational change.