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1.
The interaction of guanine, guanosine or 5′-GMP (guanosine 5′-monophosphate) with [Pd(en)(H2O)2](NO3)2 and [Pd(dapol)(H2O)2](NO3)2, where en is ethylenediamine and dapol is 2-hydroxy-1,3-propanediamine, were studied by UV-Vis, pH titration and 1H NMR. The pH titration data show that both N1 and N7 can coordinate to [Pd(en)(H2O)2]2+ or [Pd(dapol)(H2O)2]2+. The pKa of N1-H decreased to 3.7 upon coordination in guanosine and 5′-GMP complexes, which is significantly lower than that of ∼9.3 in the free ligand. In strongly acidic solution where N1-H is still protonated, only N7 coordinates to the metal ion, but as the pH increases to pH ∼3, 1H NMR shows that both N7-only and N1-only coordinated species exist. At pH 4-5, both N1-only and N1,N7-bridged coordination to Pd(II) complexes are found for guanosine and 5′-GMP. The latter form cyclic tetrameric complexes, [Pd(diamine)(μ-N1,N7-Guo]44+ and [Pd(diamine)(μ-N1,N7-5′-GMP)]4Hx(4−x)−, (x=2,1, or 0) with either [Pd(en)(H2O)2](NO3)2 or [Pd(dapol)(H2O)2](NO3)2. The pH titration data and 1H NMR data agree well with the exception that the species distribution diagrams show the initial formation of the N1-only and N1,N7-bridged complexes to occur at somewhat higher pH than do the NMR data. This is due to a concentration difference in the two sets of data. 相似文献
2.
Gisbert Grossmann Kim A. Burkov Lubov A. Myund Claudia Verwey 《Inorganica chimica acta》2004,357(3):797-808
Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H6L) have been studied by dilatometric, potentiometric and 31P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M+ + HL5− ? MHL4− and M+ + L6− ? ML5−, respectively, were determined: logKNaHL=1.08(0.07), logKKHL=0.86(0.08), logKNaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pKa5app and pKa6app. Correction formulae were derived to convert actual dissociation constants pKa into apparent dissociation constants pKaapp (or vice versa). The actual dissociation constants were found: pKa5(H2L4− ? H+ + HL5−)=7.47(0.03) and pKa6(HL5− ? H+ + L6−)=14.1(0.1). The anisotropy of 31P chemical shifts of salts MnH6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts. 相似文献
3.
The synthetic octapeptide peptide T (ASTTTNYT) has been shown to interfere with binding of the HIV-1 envelope glycoprotein gp120 to the chemokine receptor R5, thus preventing viral infection. This study investigated the degree of conformational order of two analogs of peptide T, one biologically active (D-Ala peptide T amide) and one inactive (D-Ala, D-Tyr peptide T amide) using nuclear magnetic resonance (NMR) spectroscopy in an aqueous environment, both in solution and in the frozen solid state. Standard solution NMR techniques such as DQFCOSY, HMQC, ROESY and inversion recovery measurements have been utilized to characterize these peptides. Solid state NMR experiments were likewise employed to study the peptides in a frozen glycerol:water mixture. The NMR results indicate that the monomeric form of both peptide T analogs have considerable conformational heterogeneity. Solid state NMR studies indicate aggregation of D-Ala peptide T, possibly into a beta-sheet structure, at concentrations higher than 10 mM. 相似文献
4.
Wszelaka-Rylik M Witkiewicz-Kucharczyk A Wójcik J Bal W 《Journal of inorganic biochemistry》2007,101(5):758-763
Diadenosine 5',5'-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo. 相似文献
5.
J. -P. Issartel A. Dupuis C. Moral J. -L. Girardet 《European biophysics journal : EBJ》1991,20(2):115-126
Different kinds of nucleotide binding enzymes are sensitive to fluoroberyllate complexes (BeF.) and fluoroaluminate complexes (AlFy). It has been hypothesized that the effects of these fluorometals are related to the generation at a nucleotide binding site of a pseudo nucleoside triphosphate, consisting of a fluorometal moiety bound to the phosphate group of a molecule of nucleoside diphosphate (Bigay et al. 1985; Lunardi et al. 1985). In order to establish whether ternary complexes comprising ADP, beryllium and fluoride can exist in slightly alkaline solution in the absence of enzyme, we have carried out a multinuclear (31P, 9Be and t9F) NMR study. In preliminary experiments, pyrophosphate (PPi) was substituted for ADP and taken as a simpler analog of nucleoside diphosphate. In the absence of fluoride, three types of PPi-Be complexes were generated: two of these were bidentate chelates with either one or two pyrophosphate molecules bound per beryllium; the third one was a monodentate complex. It is probable that the same types of combination exist between the polyphosphate chain of ADP and Be. In the presence of fluoride, both ADP and PPi combined with beryllium to form ternary complexes. These complexes consisted of monofluoroberyllate(-BeF) or difluoroberyllate (-BeF,) bound to the two phosphates of one molecule of ADP or PPi as a bidentate chelate. We failed to observe the formation of complexes between ADP and trifluoroberyllate (-BeF3). The relevance of this study to the biological effects of fluoride and beryllium on various enzymic reactions is discussed.Abbreviations PPi
pyrophosphate
- AMP
adenosine -5-monophos-phate
- ADP
adenosine- 5-diphosphate
- ADPS
adenosine-5-O-(2-thiodiphosphate)
- Ap2A
P1,p2-di(adenosine-5)pyrophosphate
- F1-ATPase
catalytic sector (soluble) of the beef heart mitochondrial ATPase complex
-
Tris
tris(hydroxymethyl)aminomethane
Offprint requests to: J.-L. Girardet 相似文献
6.
Rajagopalan S Chow C Raghunathan V Fry CG Cavagnero S 《Journal of biomolecular NMR》2004,29(4):505-516
Due to the inherent complexity of the natural biological environment, most studies on polypeptides, proteins and nucleic acids have so far been performed in vitro, away from physiologically relevant conditions. Nuclear magnetic resonance is an ideal technique to extend the in vitro analysis of simple model systems to the more complex biological context. This work shows how diffusion-based spectroscopic selection can be combined with isotopic labeling to tackle and optimize the NMR analysis of specific macromolecules in multicomponent mixtures. Typical media include cell-free systems containing overexpressed proteins, lysates and proteolytic mixtures. We present a few variants of diffusion-edited HSQC pulse sequences for the selective spectroscopic detection of protein and polypeptide resonances within complex mixtures containing undesired species of smaller molecular weight. Due to diffusion-based filtering, peak intensities of fast diffusing small molecules are attenuated more than peaks due to large molecules. The basic sequence, denoted as PFGSTE-HSQC, combines translational diffusion-ordering with two dimensional heteronuclear single quantum correlation spectroscopy. The GCSTE-HSQC and BPPSTE-HSQC sequences include bipolar gradients and are therefore suitable for both diffusion-based filtering and determination of diffusion coefficients of individual mixture components. Practical applications range from protein stability/folding investigations in physiologically relevant contexts to prescreening of tertiary fold and resonance assignments in structural genomics studies. A few applications of diffusion-edited HSQC to an E. coli cell lysate containing the (15)N-labeled B domain of streptococcal protein G (GB1), and to a (15)N-labeled N-acetylglycine/apomyoglobin mixture are presented. In addition, we provide specific guidelines for experimental setup and parameter optimization. 相似文献
7.
The hydrogen-bond network in mono-altro-beta-cyclodextrin and in its inclusion complex with adamantane-1-carboxylic acid were investigated by (1)H NMR spectroscopy using the chemical shifts, temperature coefficients and vicinal coupling constants of the exchangeable hydroxy protons. The chemical shifts of the 3-OH signals indicated that the hydrogen-bond network between the 2-OH and 3-OH groups was disturbed not only on each side of the altrose residue, but also along the whole dextrin chain. Upon addition of adamantane-1-carboxylic acid, altrose underwent a conformational change from the (1)C(4) to the (O)S(2) form, allowing a more continuous belt of hydrogen bonding, as evidenced by the downfield shift experienced by the 3-OH proton signals of the glucose residues. 相似文献
8.
Leovac VM Joksović MD Divjaković V Jovanović LS Saranović Z Pevec A 《Journal of inorganic biochemistry》2007,101(7):1094-1097
A copper(II) complex with the pyridoxal-aminoguanidine (PL-AG) Schiff base adduct, as an organic compound of the very potent biological activity and promising pharmacological importance in the treatment of diabetic complications, has been prepared and characterized. The X-ray structural analysis of the [CuCl2(PL-AG)] complex showed that it has a distorted pseudo-square-pyramidal (4+1) structure with the tridentate ONN Schiff base in the equatorial plane, with the Cu-O(1), Cu-N(1) and Cu-N(3) bond lengths of 1.917(2)A, 1.930(2)A and 1.984(2)A, respectively. The bond length of the equatorial Cu-Cl(1) is 2.279(1)A, while that of the apical Cu-Cl(2) is 2.792(1)A. Pyridoxal fragment is coordinated in its zwitterionic form. In addition to the X-ray structural analysis, the complex was characterized by IR spectrometric, conductometric and magnetic techniques, and the ligand itself by IR, 1H and 13C NMR spectra. 相似文献
9.
Sebastian Meier Bent O. Petersen Jens
. Duus Ole W. Srensen 《Carbohydrate research》2009,344(16):2274-2278
The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of 13C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the 1H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution. 相似文献
10.
《FEBS letters》1994,350(2-3):275-280
Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with 1H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pKa values of which range from 3.6 to 7.4. These pKa values were assigned to individual histidine residues from NMR studies on a series of His → Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding. 相似文献
11.
Sorin A. Lusceac Michael R. Vogel Claudia R. Herbers 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(1):41-48
2H NMR spin-lattice relaxation and line-shape analyses are performed to study the temperature-dependent dynamics of water in the hydration shells of myoglobin, elastin, and collagen. The results show that the dynamical behaviors of the hydration waters are similar for these proteins when using comparable hydration levels of h = 0.25–0.43. Since water dynamics is characterized by strongly nonexponential correlation functions, we use a Cole–Cole spectral density for spin-lattice relaxation analysis, leading to correlation times, which are in nice agreement with results for the main dielectric relaxation process observed for various proteins in the literature. The temperature dependence can roughly be described by an Arrhenius law, with the possibility of a weak crossover in the vicinity of 220 K. Near ambient temperatures, the results substantially depend on the exact shape of the spectral density so that deviations from an Arrhenius behavior cannot be excluded in the high-temperature regime. However, for the studied proteins, the data give no evidence for the existence of a sharp fragile-to-strong transition reported for lysozyme at about 220 K. Line-shape analysis reveals that the mechanism for the rotational motion of hydration waters changes in the vicinity of 220 K. For myoglobin, we observe an isotropic motion at high temperatures and an anisotropic large-amplitude motion at low temperatures. Both mechanisms coexist in the vicinity of 220 K. 13C CP MAS spectra show that hydration results in enhanced elastin dynamics at ambient temperatures, where the enhancement varies among different amino acids. Upon cooling, the enhanced mobility decreases. Comparison of 2H and 13C NMR data reveals that the observed protein dynamics is slower than the water dynamics. 相似文献
12.
Soong-Hoon Kim Keith L. Constantine Gerald J. Duke Valentina Goldfarb John T. Hunt Stephen Johnson Kevin Kish Herbert E. Klei Patricia A. McDonnell William J. Metzler Luciano Mueller Michael A. Poss Craig R. Fairchild Rajeev S. Bhide 《Bioorganic & medicinal chemistry letters》2013,23(14):4107-4111
The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11 μM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47 μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg2+. In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca2+ and Mg2+ are indicative of a highly dynamic process in the active site for the HDHD4/Mg2+/3 complex. Possible explanations for this observation are discussed. 相似文献
13.
Innokentiy Maslennikov Martin Krupa Christopher Dickson Luis Esquivies Katherine Blain Georgia Kefala Senyon Choe Witek Kwiatkowski 《Journal of structural and functional genomics》2009,10(1):25-35
Abstract Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane
proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of
an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used
in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent
complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably,
this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one
particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein
oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties
through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for
solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact
protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating
PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore
more effective than centrifugal ultrafiltration devices. 相似文献
14.
Erythritol was chosen to study the interactions between metal ions and carbohydrates. FTIR spectroscopy results indicate that a EuCl3-erythritol complex different from a previously reported one was obtained. The crystal structure of EuCl3-erythritol complex, 2EuCl3.2C4H10O4.7H2O, Mr=443.49, a=13.846(3) A , b=7.4983(15) A, c=14.140(3) A, beta=116.39(3) degrees, V=1315.1(5) A(3), Z=4, mu=5.394 mm(-1) and R=0.0395 for 2965 observed reflections and 143 parameters, was determined. Characteristic of this complex is the presence of binuclear europium ions with different coordination structures. One Eu3+ ion is nine-coordinated, with five Eu-O bonds from water molecules, and four from hydroxyl groups of two erythritol molecules and another Eu3+ is eight-coordinated with two water molecules, two chloride ions, and four hydroxyl groups from two erythritol molecules. Erythritol provides two hydroxyl groups to one lanthanide ion and the other two to another rare earth ion. The OH, CO stretching and other vibrations are shifted in the IR spectra of the complexes and the results are consistent with the crystal structure. 相似文献
15.
16.
Biancamaria Farina Annarita Del Gatto Daniela Comegna Sonia Di Gaetano Domenica Capasso Carla Isernia Michele Saviano Roberto Fattorusso Laura Zaccaro Luigi Russo 《Journal of peptide science》2019,25(5)
Integrins are heterodimeric cell‐surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin‐inhibitor drugs have been developed for integrin‐based medical applications, the structural determinants modulating integrin‐ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C‐terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for αvβ3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of αvβ3 by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of αvβ3 integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance 15N and 13C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner. 相似文献
17.
Marc Lecouvey Céline Frochot Laurent Miclo Piotr Orlewski Michel Marraud Jean-Luc Gaillard Manh Thong Cung Régis Vanderesse 《International journal of peptide research and therapeutics》1997,4(4-6):359-364
Summary The conformation of a benzodiazepine-like decapeptide corresponding to the YLGYLEQLLR fragment of a casein has been examined
in a sodium dodecyl sulfate micellar medium using circular dichroism, two-dimensional1H NMR spectroscopy and restrained molecular dynamics simulation. The decapeptide adopts an amphipathic 310-helicoid structure in which the E6...R10 ionic bridge stabilizes the C-terminus. 相似文献
18.
19.
Yuanyuan Yang Yiluan Ding Chen Zhou Yi Wen Naixia Zhang 《Protein science : a publication of the Protein Society》2019,28(9):1606-1619
Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia‐inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N‐terminal zinc‐finger ubiquitin binding domain (ZnF‐UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF‐UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF‐UBP domain and investigated its binding properties with mono‐ubiquitin and poly‐ubiquitin (K48‐linked di‐ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF‐UBP domain forms a spherically shaped fold consisting of a central β‐sheet with either one α‐helix or two α‐helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF‐UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF‐UBP's ubiquitin binding capacity was revealed by NMR data‐driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF‐UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di‐glycine motif and the conserved and non‐conserved residues of USP20 ZnF‐UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF‐UBP domain might have a physiological role unrelated to its ubiquitin binding capacity. 相似文献
20.
The heptadecapeptides bombolitin I and bombolitin III are two members of a series of biologically active peptides postulated to be membrane active. In order to understand the effects of the membrane on the secondary structure of the peptides, we have carried out the conformational characterization of bombolitins I and III in the presence of SDS micelles using circular dichroism, nuclear magnetic resonance, and computer simulations. The characteristic bands in the circular dichroism spectra indicate an alpha-helix content of approximately 60% in bombolitin III and 70% in bombolitin I. The observation of NOE's quite distinctive for such secondary structure strongly supports the CD results. The conformational preferences of the two bombolitins derived from CD and NMR were then energetically refined with molecular dynamics simulations. The results from the spectroscopic examination were utilized as input for the simulations, the CD results for generation of the initial structure, and the NOE's as constraints during the simulations. The results from the different techniques employed are in complete agreement. 相似文献