Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2)

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.     

Independence of metal binding between tandem Cys2His2 zinc finger domains.

Most Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations.     

Physiological levels of glutathione enhance Zn(II) binding by a Cys4 zinc finger

Using fluorescence and UV-vis spectroscopies and mass spectrometry, we demonstrated that the presence of physiological levels of reduced glutathione enhances the binding of Zn(II) to XPAzf, a Cys4 zinc finger peptide derived from the XPA protein, by means of formation of a ternary complex of a general formula ZnXPAzf[GSH]. Similar complexes were also indicated by ESI-MS for isostructural Co(II)- and Cd(II)-substituted XPAzf. The observed enhancement of the Zn(II) binding to XPAzf by a factor of 50 over the physiological range of GSH concentrations of 1-20 mM corresponds to a dissociation constant of GSH from the ZnXPAzf[GSH] complex of 0.05 μM. This effect may account for an apparent discrepancy between relatively low Zn(II) binding constants measured in vitro for many zinc fingers, and the requirement of tight Zn(II) binding enforced by intracellular zinc buffering by the thionein/metallothionein couple.     

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling

Free zinc ions are potent effectors of proteins. Their tightly controlled fluctuations ("zinc signals") in the picomolar range of concentrations modulate cellular signaling pathways. Sulfur (cysteine) donors generate redox-active coordination environments in proteins for the redox-inert zinc ion and make it possible for redox signals to induce zinc signals. Amplitudes of zinc signals are determined by the cellular zinc buffering capacity, which itself is redox-sensitive. In part by interfering with zinc and redox buffering, reactive species, drugs, toxins, and metal ions can elicit zinc signals that initiate physiological and pathobiochemical changes or lead to cellular injury when free zinc ions are sustained at higher concentrations. These interactions establish redox-inert zinc as an important factor in redox signaling. At the center of zinc/redox signaling are the zinc/thiolate clusters of metallothionein. They can transduce zinc and redox signals and thereby attenuate or amplify these signals.     

Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals

Zinc(II) ions are essential for all forms of life. In humans, they have catalytic and structural functions in an estimated 3,000 zinc proteins. In addition, they interact with proteins transiently when they regulate proteins or when proteins regulate cellular zinc re-distribution. As yet, these types of zinc proteins have been explored poorly. Therefore the number of zinc/protein interactions is potentially larger than that given by the above estimate. Confronted with such a wide range of functions, which affect virtually all aspects of cellular physiology, investigators have begun to elucidate the molecular mechanisms of cellular homeostatic control of zinc, especially the functions of transporter, sensor, and trafficking proteins, such as metallothioneins, in providing the correct amounts of zinc ions for the synthesis of zinc metalloproteins. The sulfur-containing amino acid cysteine in proteins has an important role in the cellular mobility of zinc ions. Sulfur-coordination environments provide sufficiently strong interactions with zinc ions; they can undergo fast ligand-exchange; and they can serve as molecular redox switches for zinc binding and release. For the cellular functions of zinc, the free zinc ion concentrations (zinc potentials, pZn = −log[Zn²⁺]) and the zinc buffering capacity are critically important parameters that need to be defined quantitatively. In the cytoplasm, free zinc ions are kept at picomolar concentrations as a minute fraction of the few hundred micromolar concentrations of total cellular zinc. However, zinc ion concentrations can fluctuate under various conditions. Zinc ions released intracellularly from the zinc/thiolate clusters of metallothioneins or secreted from specialized organelles are potent effectors of proteins and are considered zinc signals. The cellular zinc buffering capacity determines the threshold between physiological and pathophysiological actions of zinc ions. When drugs, toxins, other transition metal ions or reactive compounds compromise zinc buffering, large zinc ion fluctuations can injure cells through effects on redox biology and interactions of zinc ions with proteins that are normally not targeted.

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

High-resolution solution structure of the double Cys2His2 zinc finger from the human enhancer binding protein MBP-1.

The high-resolution three-dimensional structure of a synthetic 57-residue peptide comprising the double zinc finger of the human enhancer binding protein MBP-1 has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1280 experimental restraints. A total of 30 simulated annealing structures were calculated. The backbone atomic root-mean-square distributions about the mean coordinate positions are 0.32 and 0.33 A for the N- and C-terminal fingers, respectively, and the corresponding values for all atoms, excluding disordered surface side chains, are 0.36 and 0.40 A. Each finger comprises an irregular antiparallel sheet and a helix, with the zinc tetrahedrally coordinated to two cysteines and two histidines. The overall structure is nonglobular in nature, and the angle between the long axes of the helices is 47 +/- 5 degrees. The long axis of the antiparallel sheet in the N-terminal finger is approximately parallel to that of the helix in the C-terminal finger. Comparison of this structure with the X-ray structure of the Zif-268 triple finger complexed with DNA indicates that the relative orientation of the individual zinc fingers is clearly distinct in the two cases. This difference can be attributed to the presence of a long Lys side chain in the C-terminal finger of MBP-1 at position 40, instead of a short Ala or Ser side chain at the equivalent position in Zif-268. This finding suggests that different contacts may be involved in the binding of the zinc fingers of MBP-1 and Zif-268 to DNA, consistent with the findings from methylation interference experiments that the two fingers of MBP-1 contact 10 base pairs, while the three fingers of Zif-268 contact only 9 base pairs.     

Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method

The C₂H₂ zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template. Following a successful selection, multiple zinc-finger clones are typically recovered that share similarities in the sequences of their DNA-recognition helices. In principle, each of the clones isolated from a selection is a candidate for assembly into a larger multi-finger protein, but to date a high-throughput method for identifying the most specific candidates for incorporation into a final multi-finger protein has not been available. Here we describe the development of a specificity profiling system that facilitates rapid and inexpensive characterization of engineered zinc-finger modules. Moreover, we demonstrate that specificity data collected using this system can be employed to rationally design zinc fingers with improved DNA-binding specificities.     

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracelular erythrocruorins (Ers), ∼ 3.5 MDa, comprized of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions forLumbricus andRiftia assemblies and their globin and linker subassemblies, based on theLumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses ofLumbricus andRiftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of ∼ 9% due to the breadth of the mass distributions. TheLumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 ± 0.481 MDa.     

Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.     

Kinetics of an enzyme-catalyzed reaction measured by electrospray ionization mass spectrometry using a simple rapid mixing attachment

Mass spectrometry offers a potential means of measuring virtually all enzyme-catalyzed reactions by simultaneously measuring the concentrations of substrates, products, and intermediates where there are differences in mass between them. To perform these measurements the reaction mixture must be aged for different times and then ionized. Electrospray ionization mass spectrometry provides the most direct means of measuring these reactions. Here we describe a simple reaction mixing and ageing attachment for an electrospray ionization mass spectrometer, built from commercially available components. We have employed this device to measure the kinetics of a model reaction, namely the hydrolysis of N2-(carbobenzyloxy)-L-lysine-p-nitrophenyl ester-catalyzed by trypsin. In this way we were able to measure the kinetics of substrate depletion, product formation, and changes in both free enzyme and acyl-enzyme intermediate concentration in the approach to steady state. With this device we were able to measure reaction times down to about 640 ms.     

‘Synthetic prospecting’ using an electrospray ionisation mass spectrometry directed survey of the alkylation and arylation chemistry of [Pt2(μ-S)2(PPh3)4]

Electrospray ionisation mass spectrometry (ESI-MS) has been used as an analytical tool in a wide-ranging scoping study of the alkylation and arylation reactions of [Pt₂(μ-S)₂(PPh₃)₄]. From these experiments, the factors that influence the formation of different product species - formed by mono- or di-alkylation - are determined. If the alkylating agent is an alkyl chloride or sulfate, monoalkylation followed by dialkylation of the two sulfido groups occurs, dependent on the alkylating power of the reagent used. For example, n-butyl chloride gives solely [Pt₂(μ-S)(μ-SBu)(PPh₃)₄]⁺ while dimethyl sulfate gives [Pt₂(μ-SMe)₂(PPh₃)₄]²⁺. This species, previously unisolated is stable in the absence of good nucleophiles, but the addition of potassium iodide results in rapid conversion to [Pt₂(μ-SMe)₂(PPh₃)₃I]⁺. This iodo complex is also observed from the reaction of [Pt₂(μ-S)₂(PPh₃)₄] with excess MeI, after the initial formation of mono- and di-methylated species. In these reactions, the iodide presumably displaces a phosphine ligand, which is then quaternised by excess alkylating agent. Changing the alkylating agent to a longer chain alkyl iodide or methyl bromide decreases the rate of alkylation of the sulfide in the initially formed [Pt₂(μ-S)(μ-SR)(PPh₃)₄]⁺. Mixed-thiolate species of the type [Pt₂(μ-SMe)(μ-SR)(PPh₃)₄]²⁺ are easily generated by reaction of [Pt₂(μ-S)(μ-SR)(PPh₃)₄]⁺ with excess Me₂SO₄ and is also dependent on the avoidance of nucleophiles. Reactions towards α,ω-dialkylating agents are surveyed; the chain length is found to have a dramatic effect on the rate of the second intramolecular cyclisation process, illustrated by a competitive reactivity study involving a mixture of Br(CH₂)₄Br and Br(CH₂)₅Br; on completion of the reaction the former gives [Pt₂{μ-S(CH₂)₄S}(PPh₃)₄]²⁺ while the latter predominantly gives monoalkylated[Pt₂(μ-S){μ-S(CH₂)₅Br}(PPh₃)₄]⁺. The reactivity of o- and p-dihaloxylenes has been explored, with the reaction with p-BrCH₂C₆H₄CH₂Br giving the bridged species [(PPh₃)₄Pt₂(μ-S)(μ-SCH₂C₆H₄CH₂S)(μ-S)Pt₂(PPh₃)₄]²⁺. Arylation reactions of [Pt₂(μ-S)₂(PPh₃)₄] with halobenzenes and 2-bromoheterocyclic compounds (pyridine, thiophene) are also described.     

Estimating the affinity of protein-ligand complex from changes to the charge-state distribution of a protein in electrospray ionization mass spectrometry

The detection of low affinity interactions between proteins and ligands by biophysical methods is challenging. It is often necessary to use competition methods that are time consuming and require well characterized known binders. A mass spectrometry approach is presented for identifying low affinity protein-ligand binding which does not require direct detection of the parent protein-ligand complex but depends on characteristic changes observed in the protein mass spectrum. We observe that on titration of ligand there are characteristic ‘charge-state shifts’ which manifest as changes in the relative intensities of protein peaks that correlate with the degree of protein-ligand complex formation. We suggest that use of this phenomenon will be particularly suitable for the identification of low affinity complexes where the intensity of any complex ion would be close to noise.     

Primary structure of three cationic peptides from porcine neutrophils Sequence determination by the combined usage of electrospray ionization mass spectrometry and Edman degradation

The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.     

Overexpression of a novel chrysanthemum Cys2/His2-type zinc finger protein gene DgZFP3 confers drought tolerance in tobacco

A drought stress-responsive Cys2/His2-type zinc finger protein gene DgZFP3 was previously isolated (Liu et al., Afr J Biotechnol 11:7781–7788, 2012b) from chrysanthemum. To assess roles of DgZFP3 in plant drought stress responses, we performed gain-of-function experiment. The DgZFP3-overexpression tobacco plants showed significant drought tolerance over the wild type (WT). The transgenic lines exhibited less accumulation of H₂O₂ under drought stress, more accumulation of proline and greater activities of peroxidase (POD) and superoxide dismutase than the WT under both control conditions and drought stress. In addition, there was greater up-regulation of the ROS-related enzyme genes (NtSOD and NtPOD) and stress-related genes (NtLEA5 and NtDREB) in transgenic lines under normal or drought conditons. Thus DgZFP3 probably plays a positive regulatory role in drought stress response and has the potential to be utilized in transgenic breeding to improve drought stress tolerance in plants.     

Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (k(cat)), the Michaelis constant (K(M)), and the specificity constant (k(cat)/K(M)) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K(M), and k(cat)/K(M) values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K(M) values (0.93, 0.37, 0.027, and 0.00015 microM(-1) s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K(M) value for Xyl(5) (136 microM) was roughly twice as high as that for Xyl(6) (73 microM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed.