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The interest in mercury radioisotopes, 197mHg (t1/2 = 23.8 h) and 197gHg (t1/2 = 64.14 h), has recently been reignited by the dual diagnostic and therapeutic nature of their nuclear decays. These isotopes emit γ-rays suitable for single photon emission computed tomography imaging and Auger electrons which can be exploited for treating small and metastatic tumors. However, the clinical utilization of 197m/gHg radionuclides is obstructed by the lack of chelators capable of securely binding them to tumor-seeking vectors. This work aims to address this challenge by investigating a series of chemically tailored macrocyclic platforms with sulfur-containing side arms, namely, 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO3S), and 1,7-bis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,10-diacetic acid (DO2A2S). 1,4,7,10-Tetrazacyclododecane-1,4,7,10-tetracetic acid (DOTA), the widest explored chelator in nuclear medicine, and the nonfunctionalized backbone 1,4,7,10-tetrazacyclododecane (cyclen) were considered as well to shed light on the role of the sulfanyl arms in the metal coordination. To this purpose, a comprehensive experimental and theoretical study encompassing aqueous coordination chemistry investigations through potentiometry, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and density functional theory (DFT) calculations, as well as concentration- and temperature-dependent [197m/gHg]Hg2+ radiolabeling and in vitro stability assays in human serum was conducted. The obtained results reveal that the investigated chelators rapidly complex Hg2+ in aqueous media, forming extremely thermodynamically stable 1:1 metal-to-ligand complexes with superior stabilities compared to those of DOTA or cyclen. These complexes exhibited 6- to 8-fold coordination environments, with donors statically bound to the metal center, as evidenced by the presence of 1H-199Hg spin-spin coupling via NMR. A similar octacoordinated environment was also found for DOTA in both solution and solid state, but in this case, multiple slowly exchanging conformers were detected at ambient temperature. The sulfur-rich ligands quantitatively incorporate cyclotron-produced [197m/gHg]Hg2+ under relatively mild reaction conditions (pH = 7 and T = 50 °C), with the resulting radioactive complexes exhibiting decent stability in human serum (up to 75% after 24 h). By developing viable chelators and understanding the impact of structural modifications, our research addresses the scarcity of suitable chelating agents for 197m/gHg, offering promise for its future in vivo application as a theranostic Auger-emitter radiometal.
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Ciclamos , Compostos Macrocíclicos , Humanos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/síntese química , Mercúrio/química , Enxofre/química , Radioisótopos/química , Estrutura Molecular , Elétrons , Complexos de Coordenação/química , Complexos de Coordenação/síntese química , Compostos Heterocíclicos/química , Nanomedicina TeranósticaRESUMO
Arsenic is highly toxic and a significant threat to human health, but certain bacteria have developed defense mechanisms initiated by AsIII binding to AsIII-sensing proteins of the ArsR family. The transcriptional regulator AfArsR responds to AsIII and SbIII by coordinating the metalloids with three cysteines, located in a short sequence of the same monomer chain. Here, we characterize the binding of AsIII and HgII to a model peptide encompassing this fragment of the protein via solution equilibrium and spectroscopic/spectrometric techniques (pH potentiometry, UV, CD, NMR, PAC, EXAFS, and ESI-MS) combined with DFT calculations and MD simulations. Coordination of AsIII changes the peptide structure from a random-coil to a well-defined structure of the complex. A trigonal pyramidal AsS3 binding site is formed with almost exactly the same structure as observed in the crystal structure of the native protein, implying that the peptide possesses all of the features required to mimic the AsIII recognition and response selectivity of AfArsR. Contrary to this, binding of HgII to the peptide does not lead to a well-defined structure of the peptide, and the atoms near the metal binding site are displaced and reoriented in the HgII model. Our model study suggests that structural organization of the metal site by the inducer ion is a key element in the mechanism of the metalloid-selective recognition of this protein.
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Arsênio , Arsênio/química , Arsênio/metabolismo , Sítios de Ligação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Metaloides/química , Metaloides/metabolismo , Teoria da Densidade Funcional , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
Thiolate containing mercury(II) complexes of the general formula [Hg(SR) n ] 2 - n have been of great interest since the toxicity of mercury was recognized. 199Hg nuclear magnetic resonance spectroscopy (NMR) is a powerful tool for characterization of mercury complexes. In this work, the Hg shielding constants in a series of [Hg(SR) n ] 2 - n complexes are therefore investigated computationally with particular emphasis on their geometry dependence. Geometry optimizations and NMR chemical shift calculations are performed at the density functional theory (DFT) level with both the zeroth-order regular approximation (ZORA) and four-component relativistic methods. The four exchange-correlation (XC) functionals PBE0, PBE, B3LYP, and BLYP are used in combination with either Dyall's Gaussian-type (GTO) or Slater-type orbitals (STOs) basis sets. Comparing ZORA and four-component calculations, one observes that the calculated shielding constants for a given molecular geometry have a constant difference of â¼ 1070 ppm. This confirms that ZORA is an acceptable relativistic method to compute NMR chemical shifts. The combinations of four-component/PBE0/v3z and ZORA/PBE0/QZ4P are applied to explore the geometry dependence of the isotropic shielding. For a given coordination number, the distance between mercury and sulfur is the key factor affecting the shielding constant, while changes in bond and dihedral angles and even different side groups have relatively little impact.
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ß-Lactamases grant resistance to bacteria against ß-lactam antibiotics. The active center of TEM-1 ß-lactamase accommodates a Ser-Xaa-Xaa-Lys motif. TEM-1 ß-lactamase is not a metalloenzyme but it possesses several putative metal ion binding sites. The sites composed of His residue pairs chelate borderline transition metal ions such as Ni(II). In addition, there are many sulfur-containing donor groups that can coordinate soft metal ions such as Hg(II). Cd(II) may bind to both types of the above listed donor groups. No significant change was observed in the circular dichroism spectra of TEM-1 ß-lactamase on increasing the metal ion content of the samples, with the exception of Hg(II) inducing a small change in the secondary structure of the protein. A weak nonspecific binding of Hg(II) was proven by mass spectrometry and 119m Hg perturbed angular correlation spectroscopy. The hydrolytic process of ampicillin catalyzed by TEM-1 ß-lactamase was described by the kinetic analysis of the set of full catalytic progress curves, where the slow, yet observable conversion of the primary reaction product into a second one, identified as ampilloic acid by mass spectrometry, needed also to be considered in the applied model. Ni(II) and Cd(II) slightly promoted the catalytic activity of the enzyme while Hg(II) exerted a noticeable inhibitory effect. Hg(II) and Ni(II), applied at 10 µM concentration, inhibited the growth of E. coli BL21(DE3) in M9 minimal medium in the absence of ampicillin, but addition of the antibiotic could neutralize this toxic effect by complexing the metal ions.
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Cádmio , Mercúrio , Cádmio/farmacologia , Escherichia coli/metabolismo , Hidrólise , Cinética , beta-Lactamases/química , Ampicilina/farmacologia , Mercúrio/farmacologia , Catálise , ÍonsRESUMO
With this work we first test various DFT functionals against CCSD(T) for calculation of EFGs at the position of Cd(II) in a very small model system, Cd(SCH3)2. Moreover, the available basis sets in ADF are tested in terms of basis set convergence, and the effect of including relativistic effects using the scalar relativistic and spin orbit ZORA Hamiltonians is explored. The results indicate that an error of up to around 10% on the calculated EFG may be expected using spin-orbit ZORA and the BHandHLYP functional with a locally dense basis set. Next, this method was applied to model systems of the CueR protein, aiming to interpret 111Ag-PAC spectroscopic data. Note that 111Ag decays to 111Cd on which the PAC data are recorded. Surprisingly, model systems truncated - as is often done - at the first C-C bond from the central Cd(II) are inadequate in size, and larger model systems must be employed to achieve reliable EFG calculations. The calculated EFGs agree well with experimental PAC data, and indicate that shortly after the nuclear decay the structure relaxes from linear two-coordinate AgS2 in the native protein, to a structure (or structures) where Cd(II) recruits additional ligands such as backbone carbonyl oxygens to achieve higher coordination number(s).
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The nanoviscosity experienced by molecules in solution may be determined through measurement of the molecular rotational correlation time, τc , for example, by fluorescence and NMR spectroscopy. With this work, we apply PAC spectroscopy to determine the rate of rotational diffusion, λ=1/τc , of a de novo designed protein, TRIL12AL16C, in solutions with viscosities, ξ, from 1.7 to 88â mPaâ s. TRIL12AL16C was selected as molecular probe because it exhibits minimal effects due to intramolecular dynamics and static line broadening, allowing for exclusive elucidation of molecular rotational diffusion. Diffusion rates determined by PAC data agree well with literature data from fluorescence and NMR spectroscopy, and scales linearly with 1/ξ in agreement with the Stokes-Einstein-Debye model. PAC experiments require only trace amounts (â¼1011 ) of probe nuclei and can be conducted over a broad range of sample temperatures and pressures. Moreover, most materials are relatively transparent to γ-rays. Thus, PAC spectroscopy could find applications under circumstances where conventional techniques cannot be applied, spanning from the physics of liquids to in-vivo biochemistry.
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Invited for the cover of this issue is the group of Artur Krezel at the University of Wroclaw in collaboration with Lars Hemmingsen at The University of Copenhagen and Eva Freisinger at the University of Zürich. The image depicts the outcomes of HgII interactions with Rad50 protein. Read the full text of the article at 10.1002/chem.202202738.
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Mercúrio , Zinco , Concentração de Íons de HidrogênioRESUMO
111Ag-perturbed angular correlation of γ-rays (PAC) spectroscopy provides information on the nuclear quadrupole interactions, and thereby on the local structure and dynamics of the silver ion binding site. Brownian rotational motion, i.e. rotational diffusion, of 111Ag-labeled molecules will significantly affect the PAC spectra. Here we illustrate this effect, by simulating 111Ag PAC spectra for 111Ag-labeled molecules with molecular masses spanning from 102 to 106 g/mol, reflecting a span from fast (small molecules) to slow (large molecules) rotational diffusion on the PAC time scale. The simulated spectra are compared to 111Ag-PAC data obtained from a pilot study involving 111Ag(I) bound to a designed chelator exhibiting fast reorientation in solution, as well as to 111Ag-labeled species formed by 111Ag(I) in human serum, exhibiting slow (or no) reorientation on the PAC time scale. The simulated and experimental data illustrate typical PAC signals that are likely to be observed in vivo, when following the fate of 111Ag-labeled compounds. Potential in vivo applications are stability studies of 111Ag-radiopharmaceuticals, dissociation studies of 111Ag from the labeled molecule followed by binding to another (bio)molecule, or binding of 111Ag-labeled probes to larger carriers such as proteins.
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Cádmio , Humanos , Projetos Piloto , Análise Espectral/métodos , Sítios de Ligação , Raios gamaRESUMO
In nature, thiolate-based systems are the primary targets of divalent mercury (HgII ) toxicity. The formation of Hg(Cys)x cores in catalytic and structural protein centers mediates mercury's toxic effects and ultimately leads to cellular damage. Multiple studies have revealed distinct HgII -thiolate coordination preferences, among which linear HgII complexes are the most commonly observed in solution at physiological pH. Trigonal or tetrahedral geometries are formed at basic pH or in tight intraprotein Cys-rich metal sites. So far, no interprotein tetrahedral HgII complex formed at neutral pH has been reported. Rad50 protein is a part of the multiprotein MRN complex, a major player in DNA damage-repair processes. Its central region consists of a conserved CXXC motif that enables dimerization of two Rad50 molecules by coordinating ZnII . Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is critical for the biological activity of the MRN. Using a series of length-differentiated peptide models of the Pyrococcus furiosus zinc hook domain, we investigated its interaction with HgII . Using UV-Vis, CD, PAC, and 199 Hgâ NMR spectroscopies as well as anisotropy decay, we discovered that all Rad50 fragments preferentially form homodimeric Hg(Hk)2 species with a distorted tetrahedral HgS4 coordination environment at physiological pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in solution. At higher HgII content, monomeric HgHk complexes with linear geometry are formed. The Hg(Cys)4 core of Rad50 is extremely stable and does not compete with cyanides, NAC, or DTT. Applying ITC, we found that the stability constant of the Rad50 Hg(Hk)2 complex is approximately three orders of magnitude higher than those of the strongest HgII complexes known to date.
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Mercúrio , Zinco , Zinco/química , Mercúrio/química , Metais , Reparo do DNA , Concentração de Íons de HidrogênioRESUMO
The complexation of MgII with adenosine 5'-triphosphate (ATP) is omnipresent in biochemical energy conversion, but is difficult to interrogate directly. Here we use the spin- 1/2 ß-emitter 31 Mg to study MgII -ATP complexation in 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) solutions using ß-radiation-detected nuclear magnetic resonance (ß-NMR). We demonstrate that (nuclear) spin-polarized 31 Mg, following ion-implantation from an accelerator beamline into EMIM-Ac, binds to ATP within its radioactive lifetime before depolarizing. The evolution of the spectra with solute concentration indicates that the implanted 31 Mg initially bind to the solvent acetate anions, whereafter they undergo dynamic exchange and form either a mono- (31 Mg-ATP) or di-nuclear (31 MgMg-ATP) complex. The chemical shift of 31 Mg-ATP is observed up-field of 31 MgMg-ATP, in accord with quantum chemical calculations. These observations constitute a crucial advance towards using ß-NMR to probe chemistry and biochemistry in solution.
Assuntos
Trifosfato de Adenosina , Magnésio , Trifosfato de Adenosina/química , Imidazóis , Espectroscopia de Ressonância Magnética/métodosRESUMO
The transcriptional regulator CueR is activated by the binding of CuI , AgI , or AuI to two cysteinates in a near-linear fashion. The C-terminal CCHHRAG sequence in Escherichia coli CueR present potential additional metal binding ligands and here we explore the effect of deleting this fragment on the binding of AgI to CueR. CD spectroscopic and ESI-MS data indicate that the high AgI -binding affinity of WT-CueR is significantly reduced in Δ7C-CueR.[111 Ag PAC spectroscopy demonstrates that the WT-CueR metal site structure (AgS2 ) is conserved, but less populated in the truncated variant. Thus, the function of the C-terminal fragment may be to stabilize the two-coordinate metal site for cognate monovalent metal ions. In a broader perspective this is an example of residues beyond the second coordination sphere affecting metal site physicochemical properties while leaving the structure unperturbed.
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Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Transativadores , Sítios de Ligação , Cobre/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ouro/química , Metais/metabolismo , Prata/química , Transativadores/metabolismoRESUMO
The kinetic recoil energy received by the daughter nucleus in a nuclear decay is often large enough to affect the structure around the nucleus in chemical systems. The coinciding element change which typically occurs in a nuclear decay may additionally incur a structural reorganization. The effects of these phenomena on chemical systems where radio-isotopes are used are often little-known or neglected because the dynamics of nuclear decay is difficult to observe experimentally. In this work, QM-MD simulations are used to investigate local fs to ps dynamics following the ß-decay of 111Ag to 111Cd in systems modelled on the metal-sensing CueR protein. An adiabatic approximation is applied, assuming that the electronic structure relaxes rapidly after the decay. PM7-MD simulations of recoil dynamics of the model systems show significant structural changes and bonding interactions that depend on the magnitude and direction of the recoil. We find that, in general, the kinetic recoil energy is rapidly distributed (<5 ps) uniformly throughout the systems in the studied scenarios.
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Accurate nuclear quadrupole moment values are essential as benchmarks for nuclear structure models and for the interpretation of experimentally determined nuclear quadrupole interactions in terms of electronic and molecular structure. Here, we present a novel route to such data by combining perturbed γ-γ angular correlation measurements on free small linear molecules, realized for the first time within this work, with state-of-the-art ab initio electronic structure calculations of the electric field gradient at the probe site. This approach, also feasible for a series of other cases, is applied to Hg and Cd halides, resulting in Q(^{199}Hg,5/2^{-})=+0.674(17) b and Q(^{111}Cd,5/2^{+})=+0.664(7) b.
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Structural characterization of aggregates and fibrils of the Aß protein is pivotal to the molecular-level elucidation of Alzheimer's disease (AD). AFM-IR spectroscopy provides nanoscale resolution, and thus allows the interrogation of individual aggregates and fibrils. During aggregation of Aß, we observed mainly disordered Aß at t=15â min, but substantial structural diversity including the co-existence of parallel and antiparallel ß-sheets within a large amorphous aggregate at t=2â hours, while fibrils exhibited the expected signature of parallel ß-sheets at t=1 week. The resonance observed for parallel ß-sheets at t=2â hours coincides with that observed for fibrils (at 1634â cm-1 ), thus indicating that fibril-like species exist within the large aggregates. Therefore, nucleation might occur within such species, in analogy to current theories of protein crystallization in which nucleation occurs within large protein clusters. Cu2+ perturbs Aß aggregation, catalysing rapid formation of amorphous aggregates with diverse secondary structure, but inhibiting fibril growth.
Assuntos
Peptídeos beta-Amiloides/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Cobre/química , Cobre/farmacologia , Humanos , Microscopia de Força Atômica , Agregados Proteicos/efeitos dos fármacos , Estrutura Secundária de Proteína , Espectrofotometria InfravermelhoRESUMO
Selectivity for monovalent metal ions is an important facet of the function of the metalloregulatory protein CueR. 111 Ag perturbed angular correlation of γ-rays (PAC) spectroscopy probes the metal site structure and the relaxation accompanying the instantaneous change from AgI to CdII upon 111 Ag radioactive decay. That is, a change from AgI , which activates transcription, to CdII , which does not. In the frozen state (-196 °C) two nuclear quadrupole interactions (NQIs) are observed; one (NQI1 ) agrees well with two coordinating thiolates and an additional longer contact to the S77 backbone carbonyl, and the other (NQI2 ) reflects that CdII has attracted additional ligand(s). At 1 °C only NQI2 is observed, demonstrating that relaxation to this structure occurs within ≈10â ns of the decay of 111 Ag. Thus, transformation from AgI to CdII rapidly disrupts the functional linear bis(thiolato)AgI metal site structure. This inherent metal site flexibility may be central to CueR function, leading to remodelling into a non-functional structure upon binding of non-cognate metal ions. In a broader perspective, 111 Ag PAC spectroscopy may be applied to probe the flexibility of protein metal sites.
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Aberrant aggregation of the Aß protein is a hallmark of Alzheimer's disease (AD), but no complete characterization of the molecular level pathogenesis has been achieved. A promising hypothesis is that dysfunction of metal ion homeostasis, and consequently, the undesired interaction of metal ions with Aß, may be central to the development of AD. Qualitatively, most data indicate that Cu(II) induces rapid self-assembly of both Aß40 and Aß42 during the initial phase of the aggregation, while at longer time scales fibrillation may occur, depending on the experimental conditions. For Aß40 and Cu(II):Aß ≤ 1, most data imply that low concentration of Aß40 favors nucleation and rapid fibril elongation, while high concentration of Aß40 favors formation of amorphous aggregates. However, there are conflicting reports on this issue. For Aß42 and Cu(II):Aß ≤ 1, there is consensus that the lag time is extended upon addition of Cu(II). For Cu(II):Aß > 1, the lag time is increased upon interaction with Cu(II), and in most cases fibrillation is not observed, presumably because Cu(II) occupies a second more solvent-exposed binding site, which is more prone to form metal ion-bridged species and cause rapid formation of non-fibrillar aggregates. The interesting N-terminally truncated Aß11-40 with high affinity for Cu(II), exhibits delay of fibrillation upon addition of 0.4 eq. Cu(II). In our view, there are still problems achieving reproducible results in this field, and we provide a shortlist of some of the pitfalls. Finally, we propose a consensus model for the effects of Cu(II) on the aggregation kinetics of Aß.
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Peptídeos beta-Amiloides/metabolismo , Cobre/metabolismo , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Humanos , Cinética , Fragmentos de Peptídeos/química , Conformação Proteica/efeitos dos fármacosRESUMO
Intracellular CuI is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. It is intriguing that HgII does not activate transcription, as bis-thiolate metal sites exhibit high affinity for HgII . Here the binding of HgII to CueR and a truncated variant, ΔC7-CueR, without the last 7 amino acids at the C-terminus including a conserved CCHH motif is explored. ESI-MS demonstrates that up to two HgII bind to CueR, while ΔC7-CueR accommodates only one HgII . 199m Hg PAC and UV absorption spectroscopy indicate HgS2 structure at both the functional and the CCHH metal site. However, at sub-equimolar concentrations of HgII at pHâ 8.0, the metal binding site displays an equilibrium between HgS2 and HgS3 , involving cysteines from both sites. We hypothesize that the C-terminal CCHH motif provides auxiliary ligands that coordinate to HgII and thereby prevents activation of transcription.
Assuntos
Cisteína/química , Proteínas de Escherichia coli/química , Mercúrio/química , Transativadores/química , Sequência de Aminoácidos , Sítios de Ligação , Cátions Bivalentes/química , Cátions Monovalentes/química , Cobre/química , Cisteína/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligantes , Mercúrio/metabolismo , Alinhamento de Sequência , Transativadores/genética , Transativadores/metabolismo , Ativação TranscricionalRESUMO
Hg2+ and Cd2+ complexation of a short hexapeptide, Ac-DCSSCY-NH2 (DY), was studied by pH-potentiometry, UV and NMR spectroscopy and fluorimetry in aqueous solutions and the Hg2+-binding ability of the ligand was also described in an immobilized form, where the peptides were anchored to a hydrophilic resin. Hg2+ was demonstrated to form a 1 : 1 complex with the ligand even at pH = 2.0 while Cd2+ coordination by the peptide takes place only above pH â¼ 3.5. Both metal ions form bis-ligand complexes by the coordination of four Cys-thiolates at ligand excess above pH â¼ 5.5 (Cd2+) and 7.0 (Hg2+). Fluorescence studies demonstrated a Hg2+ induced concentration-dependent quenching of the Tyr fluorescence until a 1 : 1 Hg2+ : DY ratio. The fluorescence emission intensity decreases linearly with the increasing Hg2+ concentration in a range of over two orders of magnitude. The fact that this occurs even in the presence of 1.0 eq. of Cd2+ per ligand reflects a complete displacement of the latter metal ion by Hg2+ from its peptide-bound form. The immobilized peptide was also shown to bind Hg2+ very efficiently even from samples at pH = 2.0. However, the existence of lower affinity binding sites was also demonstrated by binding of more than 1.0 eq. of Hg2+ per immobilized DY molecule under Hg2+-excess conditions. Experiments performed with a mixture of four metal ions, Hg2+, Cd2+, Zn2+ and Ni2+, indicate that this molecular probe may potentially be used in Hg2+-sensing systems under acidic conditions for the measurement of µM range concentrations.
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NMR spectra of Mg2+ ions in ionic liquids were recorded using a highly sensitive variant of NMR spectroscopy known as ß-NMR. The ß-NMR spectra of MgCl2 in EMIM-Ac and EMIM-DCA compare favourably with conventional NMR, and exhibit linewidths of â¼3 ppm, allowing for discrimination of species with oxygen and nitrogen coordination.
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We use a de Novo protein design strategy to demonstrate that the second coordination sphere of a metal site plays a key role in controlling coordination geometries of Cd(II)-tris-thiolate complexes. Specifically, we show that alteration of chirality within the core hydrophobic packing region of a three-stranded coiled coil (3SCC) can control the coordination number of Cd(II) by limiting steric encumbrance to the metal center. Within a specific class of 3SCCs [Ac-G-(LKALEEK) n -G-NH2], where n = 4 is TRI and n = 5 is GRAND, one L-Leu may be substituted by L-Cys to generate a planar tris-thiolate array capable of metal binding. In the native peptide containing only the L-configuration of leucine, the three-Cys ligand site leads to a mixture of 3- and 4-coordinate Cd(II). When the L-Leu above (toward the N-terminus) the tris-Cys site is substituted with D-Leu, solely a 3-coordinate structure [Cd(II)S3] was obtained. When D-Leu is located below (toward the C-terminus), a mixture of two coordination geometries, presumably Cd(II)S3O and Cd(II)S3O2, is observed, while substitution with D-Leu both above and below the tris-Cys plane yields a higher percentage of 4-coordinate Cd(II)S3O species. Thus, the use of D-amino acids around a metal's coordination sphere provides a powerful tool for controlling the properties of future designed metalloproteins.