Involvement of histidines 11, 15 and 19 in the binding of zinc to the fusogenic H5WYG peptide

The histidine‐rich GLFHAIAHFIHGGWHGLIHGWYG peptide (H5WYG) coordinates a Zn²⁺ ion and forms a stable peptide–metal complex promoting membrane fusion at physiologic pH. In our previous article titled ‘Histidine‐rich peptide: evidence for a single zinc‐binding site on H5WYG peptide that promotes membrane fusion at neutral pH’ in Journal of Mass Spectrometry (2009, 44, 81–89), tandem mass spectrometry experiments have provided evidence for the binding of a single Zn²⁺ ion to H5WYG and suggested that this binding is shared between H¹¹, H¹⁹ and probably H¹⁵ residues. To clarify the involvement of these histidine residues in the binding to the Zn²⁺ ion and especially to remove the doubt about the implication of the H¹⁵ residue, here we have used three H5WYG mutants termed H5WYGH11A, H5WYGH15A and H5WYGH19A, whose H¹¹, H¹⁵ and H¹⁹ residues were replaced with an alanine residue. The novelty introduced by these new tandem mass spectrometry experiments performed with the mutants is the demonstration that H¹⁵ is involved in the binding of the single Zn²⁺ ion and that it may even favour the setting of another Zn²⁺ ion. Thus, the three histidines H¹¹, H¹⁵ and H¹⁹ could lead to a specific structuring of H5WYG that can promote membrane fusion upon the binding of zinc. Copyright © 2009 John Wiley & Sons, Ltd.     

Unveiling VIVO2+ Binding Modes to Human Serum Albumins by an Integrated Spectroscopic–Computational Approach

Human serum albumin (HSA) is involved in the transport of metal ions and potential metallodrugs. Depending on the metal, several sites are available, among which are N-terminal (NTS) and multi-metal binding sites (MBS). Despite the large number of X-ray determinations for albumins, only one structure with Zn²⁺ is available. In this work, the binding to HSA of the V^IVO²⁺ ion was studied by an integrated approach based on spectroscopic and computational methods, which allowed the systems to be characterized even in the absence of X-ray analysis. The behavior depends on the type of albumin, defatted (HSA^d) or fatted (HSA^f). With HSA^d ‘primary’ and ‘secondary’ sites were revealed, NTS with (His3, His9, Asp13, Asp255) and MBS with (His67, His247, Asp249, Asn99 or H₂O); with increasing the ratio V^IVO²⁺/HSA^d, ‘tertiary’ sites, with one His-N and other donors (Asp/Glu-O or carbonyl-O) are populated. With HSA^f, fatty acids (FAs) cause a rotation of the subdomains IA and IIA, which results in the formation of a dinuclear ferromagnetic adduct (V^IVO)₂^D(HSA^f) with a μ_1,1-Asp249 and the binding of His247, Glu100, Glu252, and His67 or Asn99. FAs hinder also the binding of V^IVO²⁺ to the MBS.     

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+‐ATPase

Calcium ATPase is a member of the P‐type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X‐ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca²⁺ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca²⁺ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca²⁺ ion binds to site I , and then, the second Ca²⁺ ion approaches. The interactions between the second Ca²⁺ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca²⁺. Because of the electrostatic repulsion of the second Ca²⁺ and the electrostatic attraction of site II , the first bound Ca²⁺ shifts from site I to site II . Concertedly, the second Ca²⁺ binds to site I , forming a binding state with two Ca²⁺ ions, one at site I and the other at site II . Both of Glu908 and Asp800 coordinate with the two Ca²⁺ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

Influence of metal cofactors and water on the catalytic mechanism of creatininase-creatinine in aqueous solution from molecular dynamics simulation and quantum study

The reaction mechanism of creatinine-creatininase binding to form creatine as a final product has been investigated by using a combined ab initio quantum mechanical/molecular mechanical approach and classical molecular dynamics (MD) simulations. In MD simulations, an X-ray crystal structure of the creatininase/creatinine was modified for creatininase/creatinine complexes and the MD simulations were run for free creatininase and creatinine in water. MD results reveal that two X-ray water molecules can be retained in the active site as catalytic water. The binding free energy from Molecular Mechanics Poisson-Boltzmann Surface Area calculation predicted the strong binding of creatinine with Zn²⁺, Asp45 and Glu183. Two step mechanisms via Mn²⁺/Zn²⁺ (as in X-ray structure) and Zn²⁺/Zn²⁺ were proposed for water adding step and ring opening step with two catalytic waters. The pathway using synchronous transit methods with local density approximations with PWC functional for the fragment in the active region were obtained. Preferable pathway Zn²⁺/Zn²⁺ was observed due to lower activation energy in water adding step. The calculated energy in the second step for both systems were comparable with the barrier of 26.03 and 24.44 kcal/mol for Mn²⁺/Zn²⁺ and Zn²⁺/Zn²⁺, respectively.     

Joint quantum chemical and polarizable molecular mechanics investigation of formate complexes with penta- and hexahydrated Zn2+: Comparison between energetics of model bidentate,monodentate, and through-water Zn2+ binding modes and evaluation of nonadditivity effects

In order to gain an understanding of the energetics of polycoordinated Zn²⁺ binding to the formate anion (the end side chain of the Asp and Glu residues of proteins), we compare three competing binding modes in the presence of five and six water molecules: a, bidentate binding of Zn²⁺ to both formate oxygens; b, monodentate binding of Zn²⁺ to one formate oxygen; and c, through-water binding of Zn²⁺ to formate, in which the cation remains bound to its first-hydration shell waters and interacts with both formate oxygens through three water molecules. We also investigate a complex d, which is similar to c, in which formate is protonated into formic acid and one water molecule is deprotonated. The computations are carried out using the ab initio self-consistent field/MP2 with three basis sets of increasing size density functional theory, semiempirical AM1 and PM3, and the sum of interactions between fragments ab initio computed (SIBFA) molecular mechanics procedures. The summed energies of the isolated molecules making up the complexes disfavor tautomer d compared to a–c. On the other hand, the ab initio computations give the ordering of intermolecular interaction energies as d formic acid tautomer >b monodentate >a bidentate >c through-water. Whereas the first-order energy E₁ favors both inner-shell Zn²⁺ complexes with formate over the outer-shell complex, the polarization and the charge-transfer components of the second-order energy E₂ both favor the outer-shell complex over the inner-shell one, despite the increased separation between the cation and the highly polarizable formate ion. Energy balances including continuum solvation enthalpies produce an equilibration of complexes a–d. The preference favoring the monodentate complex over the bidentate one is consistent with other ab initio results for formate binding by a fully coordinated Zn²⁺ cation and with structural results from X-ray crystallography. The SIBFA results are consistent with the ab initio results, and the computed interaction energy values match the ab initio ones to within 3%. The effects of nonadditivity are analyzed in the ab initio, SIBFA, and semiempirical computations. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1379–1390, 1999     

Theoretical study of carbonic anhydrase-catalyzed hydration of co2: A brief review

Quantum mechanical calculations have been used to study the reaction mechanism of human carbonic anhydrase-catalyzed hydration of CO₂. This reaction is responsible for fast metabolism of CO₂ in the human body. For each of the reaction steps, possible catalytic effects of active site residues are examined. The pertinent results are as follows. (1) For CO₂ binding, the experimentally observed 2.5 cm^?1 frequency shift of the asymmetic stretching frequency between measurements taken in the aqueous solution and in the enzyme is reproduced in our theoretical calculations. Our results suggest that CO₂ binds to the zinc ion within the hydrophobic pocket. (2) No energy barrier is found for the nucleophilic attack from Zn²⁺?bound OH^? to C of CO₂ to form Zn²⁺?bound HCO₃^?. (3) For the internal proton transfer within zinc-bound HCO₃^?, the barrier of 35.6 kcal/mol for the direct internal proton transfer is reduced to 3.5 and 1.4 kcal/mol, respectively, when one or two water molecules are included for proton relay. (4) Displacement of Zn²⁺?bound HCO₃^? by H₂O is facilitated by the presence of the negatively charged Glu 106-Thr 199 chain and by the association and the subsequent ionization of a fifth water ligand. (5) For the intramolecular proton transfer between Zn²⁺-bound H₂O and His 64, the Zn²⁺ ion lowers the pK_a of Zn²⁺?bound water and repels the proton. His 64, or a similar proton receptor with a larger proton affinity than H₂O, functions as proton receiver; and the active site water molecules visualized by x-ray crystallography are important for the proton relay function. In summary, it is demonstrated that in order to achieve effective catalysis, a sequence of precisely coordinated catalytic events among all participating catalytic elements in the enzyme's active site is essential.     

Structural effects of Zn+2/Mg+2 ratios on crystallization characteristics and microstructure of fluorophlogopite mica-containing glass-ceramics

We report the effect of Mg⁺² substitution (by Zn⁺²) on crystallization kinetics, microstructure, thermal and mechanical properties of boroaluminosilicate glass. Zn²⁺ was selected for Mg²⁺ on the basis of similar ionic radius in six coordination system (Mg²⁺∼0.72 Å, Zn²⁺∼0.75 Å). The melt-quenched glasses with SiO₂–(1 − x) MgO–Al₂O₃–K₂O–B₂O₃–MgF₂ (BPAS)/x ZnO system, have been investigated to establish the effect of Zn⁺²/Mg⁺² ratios. It is found that the density of BPAS glass without zinc content is 2.52 g/cm³ and increased linearly on substitution of Mg²⁺ by 5–32 mol% ZnO. T_g and T_d of BPAS glass initially increased on adding 5 mol% ZnO and then decreased on further addition. From DSC study, it is found that the crystallization exotherm changes significantly in the temperature range 750–1000 °C, where different crystalline phases are formed, and the activation energy of crystallization (E_C) varies in the range of 254–388 kJ/mol. The crystalline phases formed in opaque BPAS glass-ceramic, derived by controlled heat treatment at 800 and 1050 °C (4 h), are identified as fluorophlogopite [KMg₃(AlSi₃O₁₀)F₂] mica and willemite (Zn₂SiO₄) by XRD technique, and confirmed by FTIR spectroscopy. The change of crystallization phenomena varying Zn⁺²/Mg⁺² ratios correspond to significant microstructural change. A wide range of thermal expansion (CTE) values are obtained for the BPAS glasses and corresponding glass-ceramics. CTE (50–500 °C) of BPAS glass without zinc content is 7.76 × 10⁻⁶/K, and decreased sequentially on increasing Zn⁺²/Mg⁺² ratio. The density of glass-ceramics after heating at 800 and 1050 °C increased linearly with increasing Zn⁺² substitution for Mg⁺². Microhardness of the BPAS glasses is in the range of 4.26–6.15 GPa and found to be increased to 4.58–6.78 GPa after crystallized at 1050 °C.     

A Thermodynamic Study of Zinc Ion Interaction with Bovine Carbonic Anhydrase II at Different Temperatures

A thermodynamic study of the interaction of bovine carbonic anhydrase II, CAII, with zinc ion was carried out by using isothermal titration calorimetry (ITC) at 300, 310 and 320 K in Tris buffer solutions at pH=7.5. The heats of Zn²⁺+ CAII interaction are reported and analyzed in terms of the new solvation theory. It was indicated that there are three identical and non-cooperative sites on CAII for Zn²⁺. The binding of a zinc ion is exothermic with dissociation equilibrium constants of 78.32, 95.81 and 116.70 mmol⋅L⁻¹ at 300, 310 and 320 K respectively.     

Locating Pb2+ and Zn2+ in Zinc Finger-Like Peptides Using Mass Spectrometry

The binding preferences of Pb²⁺and Zn²⁺ in doubly charged complexes with zinc finger-like 12-residue peptides (Pep), [M_n(Pep-2(n-1)H)]²⁺ have been explored using tandem mass spectrometry. The peptides were synthesized strategically by blocking the N-terminus with an acetyl group and with four cysteine and/or histidine residues in positions 2, 5, 8, and 11, arranged in different motifs: CCHH, CHCH, and CCCC. The MS² spectra of the Pb²⁺ and Zn²⁺ complexes show multiple losses of water and a single methane loss and these provide a sensitive method for locating the metal dication and so elucidating its coordination. The elimination of a methane molecule indicated the position of the metal at the Cys2 residue. Whereas lead was observed to preferentially bind to cysteine residues, zinc was found to primarily bind to histidine residues and secondarily to cysteine residues. Preferential binding of lead to cysteine is preserved in the complexes with more than one Pb²⁺. Key to the mechanism of the loss of water and methane is the metal dication withdrawing electrons from the proximal amidic nitrogen. This acidic nitrogen loses its hydrogen to an amidic oxygen situated four atoms away leading to formation of a five-member ring and the elimination of water.      

Zinc-human serum albumin association: testimony of two binding sites

The zinc cation (Zn²⁺) binding to human serum albumin (HSA) was studied using a non-equilibrium approach in order to prove two HSA binding sites. The effect of the bulk solvent pH and column temperature T on this binding and the corresponding thermodynamic data were also investigated. It appeared that the association process can be divided into two pH value ranges due to a predominant Zn²⁺ interaction with either HSA site I or site II. It was also demonstrated that the Zn²⁺ affinity for the site II was weakly affected by modifying the mobile phase pH whereas for the site I, the affinity constant increased strongly with increasing the pH of the bulk solvent.     

Molecular Dynamics Simulations of Metal Ion Binding to the His‐tag Motif

In our previous study, we have observed that the chelation of various metal ions to the His‐tag motifs mostly involves the i and i+2 His residues for Ni²⁺, Cu²⁺, Zn²⁺ and Co²+. In the present study, various 200 ps molecular dynamics simulations were further conducted to investigate the chelating pathway of various metal ions to the His‐tag motif with 6 His residues (His‐tag6) and the binding affinities of these metal binding pockets towards these metal ions. The results indicate that His‐tag6 with the chelated metal ion located in positions His(2,4) or His(3,5) exhibits the strongest affinity for Ni²+ and Cu²+.K⁺ was found to be preferred to chelate in His(1,3) and His(3,5) coordinations. However, Fe³+ was found to have higher affinity towards His(1,3) and His(2,4) binding pockets. Our results also suggest that Ni²⁺ exhibits the highest binding affinity towards His‐tag6 over the other metal ions. Most of the structural variations of the His‐tag6 motif were from the Histidyl side chains during metal ion binding. In addition, there is an inverse linear correlation between the final chelated distance and the charge/volume ratio of metal ion. There is a negative correlation between the metal binding affinity and the averaged potential energy generated from the MD simulations.     

A highly selective ratiometric fluorescent chemosensor for Zn2+ ion based on a polyimine macrocycle

A new ratiometric fluorescent chemosensor based on a polyimine macrocycle ligand 1 has been synthesized. The chemosensor can exhibit a pronounced fluorescence response and high selectivity to Zn²⁺ ion over other 15 metal ions, including Cd²⁺. Sensor 1 appears an emission peak at 370 nm. Upon the addition of Zn²⁺ ion, the typical emission peak for 1 at 370 nm is obviously quenched, but a new emission peak at around 470 nm appears and shows a large enhancement due to the formation of a 1:1 Zn²⁺-1 complex. In addition, there is a good linear relationship between the fluorescence ratio I_470nm/I_370nm and the concentration of Zn²⁺, which makes a ratiometric assay of Zn²⁺ ion possible.     

Model studies on the active site of carbonic anhydrase: Ligand properties and CO2 binding

In view of building a workable molecular model of tetraliganded zinc at the active site of carbonic anhydrase, an ab initio SCF study using pseudopotentials is performed on Zn²⁺(OH₂)_n from n = 2 to 6, Zn²⁺(NH₃)_n?1 (OH₂) for n = 2 and 4, Zn²⁺(NH₃)₂ (imidazole) (OH₂), and their ionized species involving OH^? or imidazolate, considering in particular the evolution of the properties of the ligands and of the bound cation upon increasing n and upon replacement of one ligand by another. (Comparison of NH₃ and imidazole binding was made in a full SCF calculation.) The results obtained in the tetraliganded complex confirm that zinc binding facilitates water deprotonation more than imidazole deprotonation, so as to reverse the order of their intrinsic ease of ionization. A study of the approach of CO₂ toward the active site is made in an electrostatic approximation using as models the most representative of the computed four-ligand complexes.     

Study of the binding equilibrium between Zn(II) and HSA by capillary electrophoresis-inductively coupled plasma optical emission spectrometry

A new method has been developed for following the interaction between zinc ion and human serum albumin (HSA) by capillary electrophoresis-inductively coupled plasma optical emission spectrometry. Under optimized experimental conditions, the detection limit (3σ) for free Zn²⁺ ion was found to be 1.34 μM by running 11 replicates of the reagent blank. The RSD was less than 3% and the recovery was more than 98.13%. The linear range of zinc ion concentration was between 5.1 μM and 0.3 M. The measured Zn(II)-HSA combination values of n₁ and K₁ for primary binding of Zn²⁺ to HSA were 1.09 and 2.29 × 10⁵ L mol⁻¹, respectively. The measured values of n₂ and K₂ for the non-specific binding of Zn²⁺ to HSA were 8.96 and 6.65 × 10³ L mol⁻¹, respectively. This new method allows rapid analysis of a small amount of sample, simple operation, while avoiding long periods of dialysis and eliminating interference from other metal ions. This method provides a reliable and convenient new way for studying interactions between metal ions and biomolecules.     

Zinc-induced conformational changes in the DNA-binding domain of the vitamin D receptor determined by electrospray ionization mass spectrometry

Electrospray ionization mass Spectrometry (ESI-MS) was used to measure conformational changes within the DNA-binding domain of the vitamin D receptor (VDR DBD) upon binding zinc (Zn²⁺). As increasing concentrations of Zn²⁺ were added to the VDR DBD, a gradual shift in the mass envelope to lower charge states was observed in the multiply charged spectrum. The shift in the charge states was correlated to changes observed in the far-ultraviolet circular dichroic (far-UV CD) spectrum of the protein as it was titrated with Zn²⁺. Both the multiply charged ESI and far-UV CD spectra of the Zn²⁺-titrated protein show that the binding of the first Zn²⁺ ion to the protein results in very little conformational change in the protein. The binding of a second Zn²⁺ ion resulted in a significant alteration in the structure of the protein as indicated by changes in both the multiply charged ESI and far-UV CD spectra. Much smaller changes were seen within the multiply charged ESI or far-UV CD spectra upon increasing the Zn²⁺ concentration beyond 2 mol/mol of protein. The results presented indicate that ESI-MS in combination with CD is a powerful method to measure gross conformational changes induced by the binding of metals to metalloproteins.     

Three new turn-on fluorescent sensors for the selective detection of Zn2+: Synthesis,properties and DFT studies

The design and synthesis of three new 1,8-naphthalimide-based fluorescent sensors (1–3) for the detection of Zn²⁺ in aqueous solution is described. The structural architect of these sensors contains 1,8-naphthalimide scaffold as a fluorophore attached to 2,2′-dipicolylamine (DPA) and bis(2-quinolinylmethyl)amine (DQA) receptors through an amide linkage. The addition of Zn²⁺ to the solutions of sensors (1–3) led to enhanced fluorescence intensity, ranging between 2.5 and 14 folds. At physiological pH (pH = 7.4), these sensors exhibited high selectivities for Zn²⁺ over a wide range of competing metal cations, displaying high sensitivities with a limit of detections of 120, 81.7 and 79.2 nM, respectively. This suggests that these sensors can detect chronic Zn²⁺ concentration for freshwater (>1.84 μM), designated by the U.S. Environmental Protection Agency. DFT simulations performed on the more stable stacked conformations of unbound and Zn²⁺ bounded states suggested that the latter display higher density of excited states than the unbound sensors. Moreover, the stacked conformer of sensor 3 was significantly more stable as compared to sensors 1 and 2, which was attributed to a stronger Van Der Waals (VDW) interaction between DQA and 1,8-naphthalimide. The Zn²⁺ binding leads to enhanced electronic coupling between the HOMOs and LUMOs, making excited states more populated which then undergoes geometric relaxation before emitting light and relaxing back to the ground states. The lower energy separation (5.0 eV) between the HOMO and the first Zn²⁺ d-orbital in sensor 3 as compared to sensors 1 and 2 results in enhanced density of the generated states and subsequently higher intensity upon binding with Zn²⁺.     

DNA Binding and Cleavage Activity of Zinc(II) Complex of N,N′‐Bis(2‐guanidinoethyl)‐2,6‐pyridinedicarboxamide

The interaction of zinc(II) complex of N,N′‐bis(guanidinoethyl)‐2,6‐pyridinedicarboxamide (Gua) with DNA was studied by CD spectroscopy and agarose gel electrophoresis analysis. The results indicate that the DNA binding affinity of Zn²⁺‐Gua is stronger than that of Gua and the Zn²⁺‐Gua can promote the cleavage of phosphodiester bond of supercoiled DNA under a physiological condition, which is ～10⁶ times higher than DNA natural degradation. The hydrolysis pathway was proposed as the possible mechanism for DNA cleavage promoted by the Zn²⁺‐ Gua. The acceleration is due to cooperative catalysis of the zinc cation center and the functional groups (bisguanidinium groups).     

Comparison of Ca2+ and Mg2+ binding to calmodulin. An enthalpy,entropy, and gibbs free energy analysis

A consistent set of G _B , H _B , and S _B parameters have been determined from ion specific electrode, calorimetric, and spectrophotometric studies for the binding of Ca²⁺ and Mg²⁺ to bovine calmodulin at pH=7.0 and an ionic strength I of 0.113M. A non-linear least squares analysis of calcium specific ion electrode data yields, on a molar basis, four calcium dissociation constants: 10^–7 for the first site, 10^–5 for the fourth site, and two constants between these values. Both calorimetric experiments and an indicator method provide evidence that Mg²⁺ binds to calmodulin, probably at the same sites as Ca²⁺, but with affinities about 100 times smaller: 4×10^–5 for the first site and 2×10^–3 for the fourth. Calorimetric titrations on Ca²⁺ binding to calmodulin in three buffers are consistent with 0.46 protons released upon binding at all four sites and yield an average H _B per site of 5.6 and 7.9 kJ-mol^–1 for Ca²⁺ and Mg²⁺, respectively. The entropy of the system increases by 524 and 361 J-K^–1-mol^–1 when Ca²⁺ and Mg²⁺, respectively, bind to four sites on calmodulin, i.e., the selectivity of calmodulin for Ca²⁺ is primarily derived from entropy effects. Further analysis based on elimination of the entropy term for the metal ions demonstrates that calmodulin bound to Ca²⁺ has a larger entropy than the unbound calmodulin; the opposite is true for calmodulin bound to Mg²⁺. These analyses are consistent with the hypothesis that Ca²⁺ forms tight complexes at all sites on calmodulin and that release of waters of hydration upon binding is the source of the increase of entropy in the system.     

Metal binding discrimination of the calmodulin Q41C/K75C mutant on Ca2+ and La3+

Calmodulin (CaM) is a multifunctional Ca²⁺-binding protein regulating the activity of many enzymes in response to fluctuation of the intracellular Ca²⁺ level. It has been shown that a CaM Q41C/K75C mutant (CaMSS) with a disulfide bond in the N-terminal domain exhibits greatly reduced affinity to Ca²⁺. In the present study, the experimental results revealed a unique metal binding pattern in CaMSS towards La³⁺ and Ca²⁺ separately: the mutant protein binds Ca²⁺ at site I, III and IV; however, it binds La³⁺ at site I, II and IV. A putative mechanism was proposed which is the conformation of site II (or site III) of CaMSS could be altered and thus loses its metal ion affinity in response to metal binding in the opposite terminal domain possibly through the long range domain interaction. The present work may offer new perspectives for understanding the mechanisms of specific metal ion affinity in CaM and for CaM-based protein design.     

Graphene quantum dots with Zn2+ and Ni2+ conjugates can cleave supercoiled DNA

Graphene quantum dots (GQDs), inheriting the superb property of graphene oxide, possess smaller lateral size and high biocompatibility, thus having potential in biomedical applications. We previously discovered that GQDs, combining with Cu²⁺ ions, could cleave DNA primarily through an oxidative pathway; yet, oxidative DNA cleavage is not practically preferred in biology. In this work, we explore the DNA cleavage ability of GQDs with Zn²⁺ and Ni²⁺. Zn²⁺ and Ni²⁺ alone are incapable of cleaving supercoiled DNA, but when combining with the GQDs, Zn²⁺ and Ni²⁺ exhibit DNA cleavage activity. However, the activity of these two systems is much lower than that of GQDs/Cu²⁺, and GQDs/Ni²⁺ is less active than GQDs/Zn²⁺. The functional mechanism of GQDs/Ni²⁺ and GQDs/Zn²⁺ is different from that of GQDs/Cu²⁺. The GQDs play a key role in the two systems; the redox inactive Zn²⁺ and Ni²⁺ ions assist to generate the oxidative species that eventually lead to the DNA cleavage. The current results together with our previous result indicate that GQDs together with metal ions can cleave supercoiled DNA, and their cleavage activities depend on the properties of metal ions: for redox active metal ions, metal ions play key roles, for redox inactive metal ions, GQDs are dominant.