De novo design of a stable N-terminal helical foldamer

A peptide NTH-18 was synthesized in which a N-terminal helix is stabilised by two crossed disulfide bonds to a C-terminal extension. The design was inspired by the structure of the neurotoxic peptide apamin, which has previously been used to stabilise helices in miniature enzymes. CD- and NMR-spectroscopy indicated that NTH-18 adopted a fold similar to that found in apamin. However, the arrangement of the elements of secondary structures was inverted relative to apamin; a N-terminal alpha-helix was connected by a reverse turn to a C-terminal extension of non-canonical secondary structure. NTH-18 displayed significant stability to heat and changes of pH. The high definition of the N-terminal end of the alpha-helix of NTH-18 should make this peptide a useful vehicle to stabilise alpha-helices in proteins with applications in protein engineering and molecular recognition.     

Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein

Betabellin 15D is a 64-residue, disulfide-bridged homodimer. When folded into a beta structure, the protein is predicted to have two clusters of three histidine residues, each cluster able to bind a divalent metal ion. When the protein was incubated with Cu2+, Zn2+, Co2+, or Mn2+, metal complexes of betabellin 15D were observed by electrospray-ionization mass spectrometry. The relative abundances of the ionic complexes suggested an order of affinities of Cu2+ > Zn2+ > Co2+ > Mn2+, consistent with solution-phase affinities for nitrogen- or sulfur-containing ligands. Limited proteolysis of betabellin 15D by immobilized pepsin, as measured by nanoelectrospray-ionization mass spectrometry, showed that the Phe12-Ser13 peptide bond of betabellin 15D was cleaved more slowly in the presence of Cu2+ than in its absence. Because Cu2+ has little or no effect on the catalytic rate of pepsin, the slower cleavage of the Phe12-Ser13 peptide bond may be due to its decreased accessibility caused by Cu(2+)-induced folding of betabellin 15D.     

From alpha-helix to beta-sheet--a reversible metal ion induced peptide secondary structure switch

Here we introduce a peptide model based on an alpha-helical coiled coil peptide, providing a simple system which can be used for a systematic study of the impact of different metal ions in different oxidation states on peptide secondary structure on a molecular level; histidine residues were incorporated into the heptad repeat to generate possible complexation sites for Cu2+ and Zn2+ ions.     

Conformational stability of helical peptides containing a thioamide linkage

[structure: see text] Thioxo peptide analogues of the alpha-helical peptide GCN4-p1 were synthesized and evaluated for helicity and oligomeric state. Sedimentation equilibrium and CD measurements indicate that the thioxo peptides fold into parallel alpha-helical coiled coil structures essentially identical to the native structure. This work marks the first incorporation of a thioamide linkage into the backbone of an alpha-helix and demonstrates that a thioamide linkage is compatible with positions within the helix as well as near the C-terminus.     

A survey of diamagnetic probes for copper2+ binding to the prion protein. 1H NMR solution structure of the palladium2+ bound single octarepeat

The prion protein (PrP(C)) is a copper binding cell surface glycoprotein which when misfolded causes transmissible spongiform encephalopathies. The cooperative binding of Cu2+ to an unstructured octarepeat sequence within PrP(C) causes profound folding of this region. The use of NMR to determine the solution structure of the octarepeat region of PrP with Cu2+ bound has been hampered by the paramagnetic nature of the Cu2+ ions. Using NMR we have investigated the binding of candidate diamagnetic replacement ions, to the octarepeat region of PrP. We show that Pd2+ forms diamagnetic complexes with the peptides HGGG, HGGGW and QPHGGGWGQ with 1:1 stoichiometry. The 1H NMR spectra indicate that these peptides are in slow-exchange between free and bound Pd2+ on the chemical-shift time-scale. We demonstrate that the Pd-peptide complex forms slowly with a time taken to reach half-maximal signal of 3 hours. Other candidate metal ions, Ni2+, Pt2+ and Au3+, were investigated but only the Pd2+ complexes gave resolvable 1H NMR spectra. We have determined the solution structure of the QPHGGGWGQ-Pd 1:1 complex using 71 NOE distance restraints. A backbone RMSD of 0.30 A was observed over residues 3 to 7 in the final ensemble. The co-ordinating ligands consist of the histidine imidazole side chain N epsilon, the amide N of the second and third glycines with possibly H2O as the fourth ligand. The co-ordination geometry differs markedly from that of the HGGGW-Cu crystal structure. This survey of potential replacement metal ions to Cu2+ provides insight into the metal specificity and co-ordination chemistry of the metal bound octarepeats.     

Site-specific hydration status of an amphipathic peptide in AOT reverse micelles

Reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane (IO) and water have long been used as a means to provide a confined aqueous environment for various applications. In particular, AOT reverse micelles have often been used as a template to mimic membrane-water interfaces. While earlier studies have shown that membrane-binding peptides can indeed be incorporated into the polar cavity of AOT reverse micelles where they mostly fold into an alpha-helical structure, the underlying interactions leading to the ordered conformation are however not well understood. Herein, we have used circular dichroism (CD) and infrared (IR) spectroscopies in conjunction with a local IR marker (i.e., the CN group of a non-natural amino acid, p-cyano-phenylalanine) and a global IR reporter (i.e., the amide I' band of the peptide backbone) to probe the conformation as well as the hydration status of an antimicrobial peptide, mastoparan x (MPx), in AOT reverse micelles of different water contents. Our results show that at, w0=6, MPx adopts an alpha-helical conformation with both the backbone and hydrophobic side chains mostly dehydrated, whereas its backbone becomes partially hydrated at w0=20. In addition, our results suggest that the amphipathic alpha-helix so formed orients itself in such a manner that its positively charged, lysine-rich, hydrophilic face points toward the negatively charged AOT head groups, while its hydrophobic face is directed toward the polar interior of the water pool. This picture is in marked contrast to that observed for the binding of MPx to phospholipid bilayers wherein the hydrophobic surface of the bound alpha-helix is buried deeper into the membrane interior.     

Modular nucleic acid surrogates. Solid phase synthesis of alpha-helical peptide nucleic acids (alpha PNAs)

[formula: see text] The synthesis and characterization of prototype alpha-helical peptide nucleic acid (alpha PNA) modules 1-3 as well as disulfide dimers 4 and 5 are reported. These molecules combine an alpha-helical peptidyl scaffold with well-defined nucleobase molecular recognition patterns and could serve as a basis for novel antisense and/or antigene agents. Structure assignments for these alpha PNAs were supported by MALDI-TOF mass spectrometry, and the alpha-helical nature of 4 in water was confirmed by circular dichroism (CD) spectroscopy.     

Flexible Folding: Disulfide-Containing Peptides and Proteins Choose the Pathway Depending on the Environments

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.     

化学裂解结合生物质谱对多肽二硫键的定位

二硫键是一种与多肽及蛋白质结构和功能密切相关的化学键.当多肽中存在多个半胱氨酸时,形成的二硫键可能会存在多种配对方式.快速且精准地定位多肽中多对二硫键对研究多肽的结构与功能间的关系十分重要.本文开发了一种基于化学裂解和生物质谱的新方法,对利那洛肽中3对二硫键进行了精准定位.通过解析裂解后特异肽段的二级质谱图,确定利那洛肽中3对二硫键的配对方式分别为Cys1-Cys6,Cys2-Cys10和Cys5-Cys13.该方法为二硫键的定位研究提供了新思路.     

A new fluorescent chemosensor for copper ions based on tripeptide glycyl-histidyl-lysine (GHK)

[structure: see text]. A new fluorescent chemosensor for Cu2+ ions was synthesized by modifying the tripeptide glycyl-histidyl-lysine (GHK) with 9-carbonylanthracene via the standard Fmoc solid-phase peptide synthesis method. While significant fluorescence quenching was observed from the molecule upon binding with Cu2+, addition of Fe2+, Co2+, Ni2+, and Zn2+ to the peptide solution caused a minimum fluorescence emission spectral change, indicating a high specificity of this chemosensor for Cu2+ ions. Effects of pH were also investigated.     

硫酸铜诱导烟草多酚氧化酶抗变性的研究

In order to investigate the interaction between exogenous Cu2+ and Polyphenoloxidase (PPO) from Nictiana Tobacum, the Cu2+-induced resistant-denaturation against guanidine hydrochloride(Gdn-HCl) was studied by the following enzymatic activity assay, fluorescence spectroscopy and circular dichroism (CD). The results show that the GdnHCl-induced unfolding is a two-state process with no detectable inter mediate state in the absence of exogenous Cu2+, while the GdnHCl-induced unfolding in the presence of 10.0 mmol/L exogenous Cu2+ follows a three-state transition with an intermediate state, which results from the fact that Cu2+ increases the structural stability of native PPO and its intermediate. In terms of enzymatic activities, 6 mol/L GdnHCl makes PPO lose 81.4% of its original activity after 5 min, inactivate completely after 30 min, while in the presence of 10.0 mmol/L exogenous Cu2+, only 39.4% and 75.1%, after 5 and 30 min respectively. According to the CD measurements, the relative average fractions of α-helix, anti-parallel2-sheet, 2-turn/parallel 2-sheet, aromatic residues and disulfide bond, and random coil/γ-turn are 1.1%, 3.8%, 3.3%, 7.5% and 84.3%, respectively, in the 6.0mol/L GdnHCl containing no CuSO4, but34.2%, 13.7%, 21.0%, 9.5% and 21.6%, respectively, in the same concentration of Gdn-HCl containing 10mmol/L CuSO4.     

Synthesis and X-ray crystal structure determination of the first Copper(II) complexes of tetraazamacrocycle-glyoxal condensates

Novel Cu(II) complexes CuLCl(2) (L = 1-4) have been synthesized containing the metal bound to a well-known type of tetracyclic bisaminal formed from the condensation of glyoxal and tetraazamacrocycles (1 = cyclam-glyoxal condensate, 2 = [13]aneN4-glyoxal condensate, 3 = cyclen-glyoxal condensate, 4 = isocyclam-glyoxal condensate). The four-coordinate complexes were characterized by X-ray crystallography, electronic spectroscopy, solid-state magnetic moments, and electron spin resonance spectroscopy. The tetracyclic bisaminals, although having four potential donor atoms, are bound in a cis-bidentate fashion to Cu(II) with two additional cis-chloride donors. The ligands take up folded conformations, and with the exception of ligand 4, only nonadjacent nitrogen atoms coordinate. As expected, ligand 2 in Cu(2)Cl(2) has a folded structure similar to those of the previously characterized 1 and 3. The conformation of 4 in the complex Cu(4)Cl(2) differs from 1-3 in that three nitrogens direct their lone pairs to one side of the folded tetracycle, with adjacent nitrogen atoms coordinated to Cu(II). This difference is probably caused by the presence of the more flexible seven-membered ring rather than the five- to six-membered rings in 1-3. Air oxidation of Cu(I) in the presence of 1 or 3 results in bis(mu-hydroxo) dimers as characterized by X-ray crystal structures, suggesting dioxygen binding, followed by O-O bond splitting to give the Cu(2)O(2) diamond core.     

Transient UV Raman spectroscopy finds no crossing barrier between the peptide alpha-helix and fully random coil conformation.

Transient UV resonance Raman measurements excited within the amide pi --> pi transitions of a 21 unit alpha-helical peptide has for the first time determined a lower bound for the unfolding rate of the last alpha-helical turn to form a fully random coil peptide. A 3 ns T-jump is generated with 1.9 microm laser pulses, which are absorbed by water. Subsequent 3 ns 204 nm UV pulses excite the amide Raman spectra at delay times between 3 ns and 1 ms, to monitor the peptide conformational evolution. We find approximately 180 ns relaxation times which result in a rate constant of >5 x 10(6) s(-1) for unfolding of the last alpha-helical turn. Our data are inconsistent with slow alpha-helix nuclei melting.     

吡咯并[2,1,5-cd]中氮茚酰腙衍生物的合成及Cu2+荧光探针行为

设计合成了吡咯并[2,1,5-cd]中氮茚酰腙衍生物6. 测试了其紫外光谱和荧光光谱, 研究了其对铜离子的选择性识别作用. 结果表明, 化合物6作为铜离子荧光探针, 受常见离子干扰较小, 对于铜离子有着较高的选择性和较低的检出限.     

Cyclic and hairpin peptide complexes of heme

We have synthesized and characterized a new class of heme-peptide complexes using disulfide-linked hairpin-turn and cyclic peptides and compared these to their linear analogues. The binding affinities, helicities, and mechanism of binding of linear, hairpin, and cyclic peptides to [FeIII(coproporphyrin-I)]+ have been determined. In a minimalist approach, we utilize amphiphilic peptide sequences (15-mers), where a central histidine provides heme ligation, and the hydrophobic effect is used to optimize heme-peptide complex stability. We have incorporated disulfide bridges between amphiphilic peptides to make hairpin and even cyclic peptides that bind heme extremely well, roughly 5 x 106 times more strongly than histidine itself. CD studies show that the cyclic peptide heme complexes are completely alpha-helical. NMR spectra of paramagnetic complexes of the peptides show that the 15-mer peptides bind sequentially, with an observable monopeptide, high-spin intermediate. In contrast, the cyclic peptide complexes ligate both imidazoles cooperatively to the heme, producing only a low-spin complex. Electrochemical measurements of the E1/2 of the FeIII(coproporphyrin-I)+ complexes of these peptides are all at fairly low potentials, ranging from -215 to -252 mV versus NHE at pH 7.     

On the stability of a single-turn alpha-helix: the single versus multiconformation problem

The pentapeptide Ac-HAAAH-NH2, cyclized through its imidazoles by PdII to give [Pd(en)(peptide)]2+, has recently been evaluated by 2-D NMR and simulated annealing as a single alpha-helix conformation in solution. In the present work, we have questioned this assumption by developing Pd2+ parameters for AMBER*, performing an extensive conformational search for the [Pd(en)(peptide)]2+, and deconvoluting the averaged NMR data into eight rapidly equilibrating conformations with populations ranging from 2 to 55%. None of the latter correspond to the alpha-helix, although a 3% form possesses a related structure. As a critical component of interpreting an averaged NMR spectrum in terms of a single conformation, we advise testing this assumption with a method that permits conformational deconvolution.     

Mass spectrometric analysis of the interactions between CP12, a chloroplast protein, and metal ions: a possible regulatory role within a PRK/GAPDH/CP12 complex

The small chloroplast protein CP12 plays the role of a protein linker in the assembly process of a PRK/GAPDH/CP12 complex that is involved in CO2 assimilation in photosynthetic organisms. The redox state of CP12 regulates its role as a protein linker. Only the oxidized protein, with two disulfide bonds, is active in complex formation. Several observations indicating that CP12 might bind a metal ion led us to screen the binding of different metal ions on oxidized or reduced CP12 using non-covalent electrospray ionization mass spectrometry (ESI-MS) experiments. The oxidized protein bound specifically Cu2+ and Ni2+ (Kd of 26+/-1 microM and 11+/-1 microM, respectively); other cations such as Fe2+ and Zn2+ did not bind, while cations such as Cd2+ formed non-specific adducts to CP12. Similar results were obtained for metal ions on screening with the reduced CP12. Interestingly, the present results suggest that Cu2+ catalyzes the re-formation of the disulfide bonds of the reduced CP12, leading to recovery of the fully oxidized CP12 that is then able to bind a Cu2+ ion. Finally the high similarity between CP12 and copper chaperones from Arabidopsis thaliana, as judged by hydrophobic cluster analysis, provides additional evidence for the relevance of metal binding for the in vivo situation. The findings that CP12 is able to bind a metal ion, and that Cu2+ catalyzes the oxidation of the thiol groups of CP12, are new characteristics of this protein that may prove to be important in the regulation of the assembly process of the PRK/GAPDH/CP12 complex.     

Photocontrol of DNA binding specificity of a miniature engrailed homeodomain

Control of DNA binding of HDH-3, a 18-residue polypeptide based on the recognition helix of the Q50K engrailed homeodomain, has been achieved. HDH-3 was linked to an azobenzene cross-linker through two cysteine residues in an i, i + 11 spacing. For the thermodynamically stable trans configuration of the cross-linker, the dark-adapted peptide (dad-HDH-3) adopted a mainly alpha-helical structure as judged by circular dichroism (CD) spectroscopy. After irradiation with light of 360 nm, the helical content of the peptide (irrad-HDH-3) was reduced significantly and the CD spectrum of the irradiated peptide resembled that of the largely unstructured, unalkylated peptide. Despite lacking helices-1 and -2 and the N-terminal arm of Q50K engrailed, dad-HDH-3 bound to its natural DNA target sequence TAATCC (QRE) with high affinity (K(D) = 7.5 +/- 1.3 nM). The binding affinity for the mutant DNA sequence, TAATTA (ERE), was reduced significantly (K(D) = 140 +/- 11 nM). Unlike irrad-HDH-3, which like the unalkylated parent peptide displayed only marginal DNA binding specificity, dad-HDH-3 specified base pairs 5 and 6 of QRE with an accuracy rivaling that of the intact wild-type Q50K engrailed homeodomain, making dad-HDH-3 the most specific designed DNA binding miniature homeodomain reported to date. Moreover, DNA binding affinity and specificity of HDH-3 could be controlled externally by irradiation with light.     

Synthesis,characterization and coordination chemistry of the new tetraazamacrocycle 4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-bis(methanephosphonic acid monoethyl ester) dipotassium salt

A new potentially hexadentate tetraazamacrocycle based on the cyclen skeleton has been synthesized and fully characterized. The macrocycle 4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-bis(methanephosphonic acid monoethyl ester) dipotassium salt (Me2DO2PME) contains mutually trans monoethyl ester phosphonate acid substituents on two nitrogen atoms, and trans methyl substituents on the other two nitrogen atoms. The protonation constants of this macrocycle and the stability constants of its complexes with Cu2+, Zn2+, Gd3+ and Ca2+ ions have been determined by pH potentiometric titrations. The protonation sequence of the macrocycle has been studied by 1H, 31P[1H] and 13C[1H] NMR spectroscopy: the first and second protonation steps take place at the methyl-substituted nitrogen atoms, while the third protonation involves one oxygen from a phosphonate group. Upon protonation, all the CH2 ring protons become magnetically inequivalent on the NMR time scale due to a slow conformational rearrangement, most likely occasioned by the formation of multiple hydrogen bonds within the macrocyclic ring. Me2DOPM forms neutral, mononuclear complexes with all the metals investigated. The presence of hydroxo complexes was observed for Ca2+ and Zn2+ at high pH values. Structural information on the neutral complex [Cu(Me2DO2PME)] has been obtained by a solution X-Band EPR study. It is proposed that Me2DO2PME binds Cu2+ in a distorted octahedral structure using all of its donor atoms, i.e. the four nitrogen atoms and the two phosphonate oxygen atoms. The redox chemistry of [Cu(Me2DO2PME)] in dimethyl sulfoxide and water has been studied by electrochemical measurements. Cyclic voltammetry in DMSO shows the complex to undergo a quasireversible one-electron reduction step leading to an unstable CuI species.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water coordination on the structure of glycine and zwitterionic glycine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. Here, the effect of metal ions and water on the structure of glycine is examined. The effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water on structures of Gly.Mn+(H2O)m and GlyZwitt.Mn+(H2O)m (m = 0, 2, 5) complexes have been determined theoretically by employing the hybrid B3LYP exchange-correlation functional and using extended basis sets. Selected calculations were carried out also by means of CBS-QB3 model chemistry. The interaction enthalpies, entropies, and Gibbs energies of eight complexes Gly.Mn+ (Mn+ = Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) were determined at the B3LYP density functional level of theory. The computed Gibbs energies DeltaG degrees are negative and span a rather broad energy interval (from -90 to -1100 kJ mol(-1)), meaning that the ions studied form strong complexes. The largest interaction Gibbs energy (-1076 kJ mol(-1)) was computed for the NiGly2+ complex. Calculations of the molecular structure and relative stability of the Gly.Mn+(H2O)m and GlyZwitt.Mn+(H2O)m (Mn+ = Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+; m = 0, 2, and 5) systems indicate that in the complexes with monovalent metal cations the most stable species are the NO coordinated metal cations in non-zwitterionic glycine. Divalent cations Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+ prefer coordination via the OO bifurcated bonds of the zwitterionic glycine. Stepwise addition of two and five water molecules leads to considerable changes in the relative stability of the hydrated species. Addition of two water molecules at the metal ion in both Gly.Mn+ and GlyZwitt.Mn+ complexes reduces the relative stability of metallic complexes of glycine. For Mn+ = Li+ or Na+, the addition of five water molecules does not change the relative order of stability. In the Gly.K+ complex, the solvation shell of water molecules around K+ ion has, because of the larger size of the potassium cation, a different structure with a reduced number of hydrogen-bonded contacts. This results in a net preference (by 10.3 kJ mol(-1)) of the GlyZwitt.K+H2O5 system. Addition of five water molecules to the glycine complexes containing divalent cations Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+ results in a net preference for non-zwitterionic glycine species. The computed relative Gibbs energies are quite high (-10 to -38 kJ mol(-1)), and the NO coordination is preferred in the Gly.Mn+(H2O)5 (Mn+ = Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) complexes over the OO coordination.