Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship

Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.     

Three-dimensional structure of neuropeptide k bound to dodecylphosphocholine micelles

Neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), represents the most potent and longest lasting vasodepressor and cardiomodulatory tachykinin reported thus far. NPK has been shown to have high selectivity for the NK2 receptor. Because the micelle-associated structure may be relevant to the NPK-receptor interaction, the three-dimensional structure of the NPK in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The interproton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment NPK lacks a definite secondary structure, although some turn-like elements are present in the N terminus. The structure is well-defined in the presence of dodecylphosphocholine micelles. The global fold of NPK bound to DPC micelles consists of two well-defined helices from residues 9 to 18 and residues 27 to 33 connected by a noncanonical beta turn. The N terminus of the peptide is characterized by a 3(10) helix or a series of dynamic beta turns. The conformational range of the peptide revealed by NMR and circular dichroism (CD) studies has been analyzed in terms of characteristic secondary features. The observed conformational features have been further compared to a NKA and neuropeptide gamma (NPgamma) potent endogenous agonist for the NK2 receptor.     

Iron(III) complexes: preparation, characterization, antibacterial activity and DNA-binding

Iron(III) have been combined to well known quinolones (ciprofloxacin) and some Schiff bases with the help of coordination approach. Characterization of these compounds have been done using elemental analysis, magnetic measurements, thermogravimetric analysis, IR, UV-VIS, (1)H NMR and (13)C NMR spectral investigation. Analytical studies suggest that the iron(III)-quinolone complexes assume a six-coordinated dimeric distorted octahedral geometry. All the compounds show a good antibacterial activity against broad range of bacteria like Bacillus cereus, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Serratia marcescens, whereas no significant inhibition towards growth of fungal strains like Aspergillus Niger, Aspergillus flavus and Lasiodiplodia theobromae. Analyses of all these compounds show effective sperm herring DNA inhibition.     

Unique 31P spectral response to the formation of a specific restriction enzyme-DNA complex

Protein-induced distortion is a dramatic but not universally observed feature of sequence-specific DNA interactions. This is illustrated by the crystal structures of restriction enzyme-DNA complexes: While some of these structures exhibit DNA distortion, others do not. Among the latter is Pvull endonuclease, a small enzyme that is also amenable to NMR spectroscopic studies. Here 31P NMR spectroscopy is applied to demonstrate the unique spectral response of DNA to sequence-specific protein interactions. The 31P NMR spectrum of a noncognate DNA exhibits only spectral broadening upon the addition of enzyme. However, when enzyme is added to target DNA, a number of 31P resonances shift dramatically. The magnitudes of the chemical shifts (2-3 ppm) are among the largest observed. Site-specific substitution with phosphoramidates and phosphorothioates are used analyze these effects. While such spectral features have been interpreted as indicative of DNA backbone distortions, FRET analysis indicates that this does not occur in PvuII-cognate DNA complexes in solution. The distinct 31P spectral signature observed for cognate DNA mirrors that observed for the enzyme, underscoring the unique features of cognate complex formation.     

Spectral editing and pattern recognition methods applied to high-resolution magic-angle spinning 1H nuclear magnetic resonance spectroscopy of liver tissues

Principal component analysis (PCA) has been applied to three nuclear magnetic resonance (NMR) spectral editing methods, namely, the Carr-Purcell-Meiboom-Gill spin-echo, diffusion editing, and skyline projection of a two-dimensional J-resolved spectrum, obtained from high-resolution magic-angle spinning NMR spectroscopy of liver tissues, to distinguish between control and hydrazine-treated rats. The effects of the toxin on rat liver biochemistry were directly observed and characterized by depleted levels of liver glycogen, choline, taurine, trimethylamine N-oxide, and glucose and by elevated levels of lipids and alanine. The highly unsaturated omega-3-type fatty acid was observed for the first time in hydrazine-treated rat liver. The contributions of the metabolites to the separation of control from dosed liver tissues varied depending on the type of spectral editing method used. We have shown that subtle changes in the metabolic profiles can be selectively amplified using a metabonomics approach based on the different NMR spectral editing techniques in conjunction with PCA.     

920 MHz ultra-high field NMR approaches to structural glycobiology

Although NMR spectroscopy has great potential to provide us with detailed structural information on oligosaccharides and glycoconjugates, the carbohydrate NMR analyses have been hampered by the severe spectral overlapping and the insufficiency of the conformational restraints. Recently, ultra-high field NMR spectrometers have become available for applications to structural analyses of biological macromolecules. Here we demonstrate that ultra-high fields offer not only increases in sensitivity and chemical shift dispersion but also potential benefits for providing unique information on chemical exchange and relaxation, by displaying NMR spectral data of oligosaccharide, glycoprotein, and glycolipid systems recorded at a 21.6 T magnetic field (corresponding to 920 MHz (1)H observation frequency). The ultra-high field NMR spectroscopy combined with sugar library and stable-isotope labeling approaches will open new horizons in structural glycobiology.     

Application of NMR and EPR methods to the study of RNA

The application of techniques based on magnetic resonance, specifically electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR), has provided a wealth of new information on RNA structures, as well as insights into the dynamics and function of these important biomolecules. NMR spectroscopy is very successful for determining the solution structures of small RNA domains, aptamers and ribozymes, and exploring their intramolecular dynamics and interactions with ligands. EPR-based methods have been used to map local dynamic and structural features of RNA, to explore different modes of RNA-ligand interaction, to obtain long-range structural restraints and to probe metal-ion-binding sites.     

Iron(III) complexes: Preparation,characterization, antibacterial activity and DNA-binding

Iron(III) have been combined to well known quinolones (ciprofloxacin) and some Schiff bases with the help of coordination approach. Characterization of these compounds have been done using elemental analysis, magnetic measurements, thermogravimetric analysis, IR, UV-VIS, ¹H NMR and ¹³C NMR spectral investigation. Analytical studies suggest that the iron(III)-quinolone complexes assume a six-coordinated dimeric distorted octahedral geometry. All the compounds show a good antibacterial activity against broad range of bacteria like Bacillus cereus, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Serratia marcescens, whereas no significant inhibition towards growth of fungal strains like Aspergillus Niger, Aspergillus flavus and Lasiodiplodia theobromae. Analyses of all these compounds show effective sperm herring DNA inhibition.     

Stereochemical and biochemical aspects of some organoboron complexes of sulphur donor ligands

Synthetic, structural and biochemical aspects of some organoboron complexes of sulphur containing ligands having ONS and SNNS donor systems have been described. The ligands were prepared by the condensation of 1-phenyl-1,3-butanedione, 2,4-pentanedione, diphenylethanedione, 2,3-butanedione, ethanedial and 1,4-benzenedialdehyde with 2-mercaptoaniline. The unimolar reactions between phenylboronic acid and these thio-ligands have produced Ph.B (ONS) and Ph.B. (SNNS) type of biologically active complexes. These have been characterized by elemental analysis, molecular weight determinations, and conductivity measurements. Based on UV, IR, 1H NMR, 13C NMR and 11B NMR spectral studies, a tetracoordinated state of boron has been established in all the derivatives. The ligands and their corresponding organoboron complexes have been tested in vitro against a number of pathogenic fungi and bacteria and found to possess remarkable fungicidal and bactericidal properties.     

Techniques and applications of NMR to membrane proteins (Review)

The fact that membrane proteins are notoriously difficult to analyse using standard protocols for atomic-resolution structure determination methods have motivated adaptation of these techniques to membrane protein studies as well as development of new technologies. With this motivation, liquid-state nuclear magnetic resonance (NMR) has recently been used with success for studies of peptides and membrane proteins in detergent micelles, and solid-state NMR has undergone a tremendous evolution towards characterization of membrane proteins in native membrane and oriented phospholipid bilayers. In this mini-review, we describe some of the technological challenges behind these efforts and provide examples on their use in membrane biology.     

Use of fully deuterated micelles for conformational studies of membrane proteins by high resolution 1H nuclear magnetic resonance

Micellar complexes of melittin with fully deuterated detergents have been studied by high resolution 1H nuclear magnetic resonance (NMR). The synthesis of deuterated micelles is described and it is shown that the 1H NMR spectrum of micelle-bound melittin is well resolved and suitable for detailed analysis by conventional high-resolution NMR methods. A preliminary characterization of micelle-bound melittin shows that interaction with the micelle results in different conformational and dynamic features for the hydrophobic and hydrophilic regions of the melittin amino acid sequence. The present experiments on melittin and preliminary results with other polypeptides and proteins demonstrate that in favourable cases high-resolution 1H NMR studies of the complexes formed between membrane proteins and deuterated micelles provides a viable method for conformational studies of membrane-bound proteins.     

Techniques and applications of NMR to membrane proteins

The fact that membrane proteins are notoriously difficult to analyse using standard protocols for atomic-resolution structure determination methods have motivated adaptation of these techniques to membrane protein studies as well as development of new technologies. With this motivation, liquid-state nuclear magnetic resonance (NMR) has recently been used with success for studies of peptides and membrane proteins in detergent micelles, and solid-state NMR has undergone a tremendous evolution towards characterization of membrane proteins in native membrane and oriented phospholipid bilayers. In this mini-review, we describe some of the technological challenges behind these efforts and provide examples on their use in membrane biology.     

Antibacterial activity and spectral studies of trivalent chromium, manganese, iron macrocyclic complexes derived from oxalyldihydrazide and glyoxal

A new series of complexes is synthesized by template condensation of oxalyldihydrazide and glyoxal in methanolic medium in the presence of trivalent chromium, manganese and iron salts forming complexes of the type: [M(C(8)H(8)N(8)O(4))X]X(2) where M = Cr(III), Mn(III), Fe(III) and X = Cl(-1), NO(-1)(3), CH(3)COO(-1). The complexes have been characterized with the help of elemental analyses, conductance measurements, magnetic susceptibility measurements, electronic, NMR, infrared and far infrared spectral studies. On the basis of these studies, a five coordinate square pyramidal geometry for these complexes has been proposed. The biological activities of the metal complexes were tested in vitro against a number of pathogenic bacteria and some of the complexes exhibited remarkable antibacterial activities.     

Coordination behavior and antifungicidal, antibacterial, and antifertility activities of dioxomolybdenum(VI) complexes of biologically active heterocyclic benzothiazolines

Synthesis, characterization, and structural aspects of a new class of dioxomolybdenum(VI) complexes with biologically active benzothiazolines, 2-(2-pyridyl) benzothiazoline, 2-(2-thienyl) benzothiazoline, 2-(2-furyl) benzothiazoline, 2-(3-indolyl) benzothiazoline, glyoxal benzothiazoline, biacetylbenzothiazoline, benzil benzothiazoline, and terphthaldehyde benzothiazoline have been described. The newly synthesized complexes, [MoO2(Bzt)2] and [MoO2(Bzt')] (where BztH and Bzt'H2 represent benzothazoline molecules) have been characterized by elemental analysis, conductance measurements, molecular weight determinations, and magnetic studies. Based on IR, 1H NMR, 13C NMR, and electronic spectral studies, distorted octahedral geometry with cis-MoO2 group has been indicated for the resulting complexes. Two benzothiazolines and their corresponding metal complexes were screened for their antifungicidal and antibacterial activity on several fungi and bacteria and found to be quite active in this respect. In addition, antifertility activity of representative ligands and their molybdenum complexes was also evaluated and discussed in male mice.     

Unique 31P Spectral Response to the Formation of a Specific Restriction Enzyme–DNA Complex

Protein-induced distortion is a dramatic but not universally observed feature of sequence-specific DNA interactions. This is illustrated by the crystal structures of restriction enzyme–DNA complexes: While some of these structures exhibit DNA distortion, others do not. Among the latter is PvuII endonuclease, a small enzyme that is also amenable to NMR spectroscopic studies. Here ³¹P NMR spectroscopy is applied to demonstrate the unique spectral response of DNA to sequence-specific protein interactions. The ³¹P NMR spectrum of a noncognate DNA exhibits only spectral broadening upon the addition of enzyme. However, when enzyme is added to target DNA, a number of ³¹P resonances shift dramatically. The magnitudes of the chemical shifts (2–3 ppm) are among the largest observed. Site-specific substitution with phosphoramidates and phosphorothioates are used analyze these effects. While such spectral features have been interpreted as indicative of DNA backbone distortions, FRET analysis indicates that this does not occur in PvuII-cognate DNA complexes in solution. The distinct ³¹P spectral signature observed for cognate DNA mirrors that observed for the enzyme, underscoring the unique features of cognate complex formation.     

Membrane‐induced structure of novel human tachykinin hemokinin‐1 (hHK1)

PPT‐C encoded hemokinin‐1(hHK‐1) of Homo sapiens (TGKASQFFGLM) is a structurally distinct neuropeptide among the tachykinin family that participate in the NK‐1 receptor downstream signaling processes. Subsequently, signal transduction leads to execution of various effector functions which includes aging, immunological, and central nervous system (CNS) regulatory actions. However the conformational pattern of ligand receptor binding is unclear. The three‐dimensional structure of the hemokinin‐1 in aqueous and micellar environment has been studied by one and two‐dimensional proton nuclear magnetic resonance (2D 1H‐NMR spectroscopy) and distance geometry calculations. Data shows that hemokinin‐1 was unstructured in aqueous environment; anionic detergent SDS induces α‐helix formation. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient‐COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (CYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Helical conformation is observed from residue K3‐M11. The conformational range of the peptide revealed by NMR studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared to that of Substance P potent NK1 agonist. Thus the report provides a structural insight to study hHK‐1‐NK1 interaction that is essential for hHK1 based signaling events. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 702–710, 2015.     

Complexes of Photosynthetic Redox Proteins Studied by NMR

In the photosynthetic redox chain, small electron transfer proteins shuttle electrons between the large membrane-associated redox complexes. Short-lived but specific protein:protein complexes are formed to enable fast electron transfer. Recent nuclear magnetic resonance (NMR) studies have elucidated the binding sites on plastocyanin, cytochrome c (6) and ferredoxin. Also the orientation of plastocyanin in complex with cytochrome f has been determined. Based on these results, general features that enable the formation of such transient complexes are discussed.     

Studies on biologically active complexes of cobalt(II) and nickel(II) with dithiooxamide-derived ligands.

A number of cobalt(II) and nickel(II) complexes with novel dithiooxamide ligands derived from precursors such as benzonitrile, benzosulfonamide, benzaldehyde, benzoic acid, and phenol have been synthesized and characterized on the basis of their elemental analysis molar conductance, magnetic moment, H NMR and electronic and IR spectral data. These ligands and their complexes have been screened for their antibacterial activities against bacterial species Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Tuberculi bacilli. The studies indicate that in comparison to the ligands their metal complexes are more potent and antibacterial.     

920 MHz ultra-high field NMR approaches to structural glycobiology

Although NMR spectroscopy has great potential to provide us with detailed structural information on oligosaccharides and glycoconjugates, the carbohydrate NMR analyses have been hampered by the severe spectral overlapping and the insufficiency of the conformational restraints. Recently, ultra-high field NMR spectrometers have become available for applications to structural analyses of biological macromolecules. Here we demonstrate that ultra-high fields offer not only increases in sensitivity and chemical shift dispersion but also potential benefits for providing unique information on chemical exchange and relaxation, by displaying NMR spectral data of oligosaccharide, glycoprotein, and glycolipid systems recorded at a 21.6 T magnetic field (corresponding to 920 MHz ¹H observation frequency). The ultra-high field NMR spectroscopy combined with sugar library and stable-isotope labeling approaches will open new horizons in structural glycobiology.