137 research outputs found
Design and applications of lanthanide chelating tags for pseudocontact shift NMR spectroscopy with biomacromolecules
In this review, lanthanide chelating tags and their applications to pseudocontact shift NMR spectroscopy as well as analysis of residual dipolar couplings are covered. A complete overview is presented of DOTA-derived and non-DOTA-derived lanthanide chelating tags, critical points in the design of lanthanide chelating tags as appropriate linker moieties, their stability under reductive conditions, e.g., for in-cell applications, the magnitude of the anisotropy transferred from the lanthanide chelating tag to the biomacromolecule under investigation and structural properties, as well as conformational bias of the lanthanide chelating tags are discussed. Furthermore, all DOTA-derived lanthanide chelating tags used for PCS NMR spectroscopy published to date are displayed in tabular form, including their anisotropy parameters, with all employed lanthanide ions, C; B; -Ln distances and tagging reaction conditions, i.e., the stoichiometry of lanthanide chelating tags, pH, buffer composition, temperature and reaction time. Additionally, applications of lanthanide chelating tags for pseudocontact shifts and residual dipolar couplings that have been reported for proteins, protein-protein and protein-ligand complexes, carbohydrates, carbohydrate-protein complexes, nucleic acids and nucleic acid-protein complexes are presented and critically reviewed. The vast and impressive range of applications of lanthanide chelating tags to structural investigations of biomacromolecules in solution clearly illustrates the significance of this particular field of research. The extension of the repertoire of lanthanide chelating tags from proteins to nucleic acids holds great promise for the determination of valuable structural parameters and further developments in characterizing intermolecular interactions
Charged acrylamide copolymer gels as media for weak alignment
The use of mechanically strained acrylamide/acrylate copolymers is reported as a new alignment medium for biomacromolecules. Compared to uncharged, strained polyacrylamide gels, the negative charges of the acrylamide/acrylate copolymer strongly alter the alignment tensor and lead to pronounced electroosmotic swelling. The swelling itself can be used to achieve anisotropic, mechanical strain. The method is demonstrated for the alignment of TipAS, a 17kDa antibiotic resistance protein, as well as for human ubiquitin, where alignment tensors with an AZZ,NH of up to 60Hz are achieved at a gel concentration of 2% (w/v). The alignment can be modulated by the variation of pH, ionic strength, and gel concentration. The high mechanical stability of the swollen gels makes it possible to obtain alignment at polymer concentrations of less than 1% (w/v
Application of Paramagnetic Lanthanoid Chelating Tags in NMR Spectroscopy and Their Use for the Localization of Ligands Within Biomacromolecules
The application of paramagnetic lanthanoid chelating tags for the localization of ligands within biomacromolecules enables the elucidation of binding pockets and positioning of ligands within given targets of interest, a crucial prerequisite for rational drug design. This case study consists of an overview about lanthanoid chelating tags, a description of their structural properties, the induced anisotropy as well as approaches for the localization of ligands within biomacromolecules. The localization of sulfonamide inhibitors within human carbonic anhydrase II with an accuracy of up to 0.8 Ă
over distances of 22â38 Ă
using 19F pseudocontact shift is presented as a practical example
Nonacethrene Unchained: A Cascade to Chiral Contorted Conjugated Hydrocarbon with Two sp3-Defects
We demonstrate that structurally complex carbon nanostructures can be achieved via a synthetic approach that capitalizes on a Ï-radical reaction cascade. The cascade is triggered by oxidation of a dihydro precursor of helical diradicaloid nonacethrene to give a chiral contorted polycyclic aromatic hydrocarbon named hypercethrene. In this ten-electron oxidation process, four Ï-bonds, one Ï-bond, and three six-membered rings are formed in a sequence of up to nine steps to yield a 72-carbon-atom warped framework, comprising two configurationally locked [7]helicene units, a fluorescent peropyrene unit, and two precisely installed sp3-defects. The key intermediate in this cascade is a closed nonacethrene derivative with one quaternary sp3-center, presumably formed via an electrocyclic ring closure of nonacethrene, which, when activated by oxidation, undergoes a reaction cascade analogous to the oxidative dimerization of phenalenyl to peropyrene. By controlling the amount of oxidant used, two intermediates and one side product could be isolated and fully characterized, including single-crystal X-ray diffraction analysis, and two intermediates were detected by electron paramagnetic resonance spectroscopy. In concert with density functional theory calculations, these intermediates support the proposed reaction mechanism. Compared to peropyrene, the absorption and emission of hypercethrene are slightly red-shifted on account of extended Ï-conjugation and the fluorescence quantum yield of 0.45 is decreased by a factor of âŒ2. Enantiomerically enriched hypercethrene displays circularly polarized luminescence with a brightness value of 8.3 M-1 cm-1. Our results show that reactions of graphene-based Ï-radicals-typically considered an "undefined decomposition" of non-zero-spin materials-can be well-defined and selective, and have potential to be transformed into a step-economic synthetic method toward complex carbon nanostructures
Nonacethrene Unchained: A Cascade to Chiral Contorted Conjugated Hydrocarbon with Two spÂł-Defects
We demonstrate that structurally complex carbon nanostructures can be achieved via a synthetic approach that capitalizes on a Ï-radical reaction cascade. The cascade is triggered by oxidation of a dihydro precursor of helical diradicaloid nonacethrene to give a chiral contorted polycyclic aromatic hydrocarbon named hypercethrene. In this ten-electron oxidation process, four Ï-bonds, one Ï-bond, and three six-membered rings are formed in a sequence of up to nine steps to yield a 72-carbon-atom warped framework, comprising two configurationally locked [7]helicene units, a fluorescent peropyrene unit, and two precisely installed sp; 3; -defects. The key intermediate in this cascade is a closed nonacethrene derivative with one quaternary sp; 3; -center, presumably formed via an electrocyclic ring closure of nonacethrene, which, when activated by oxidation, undergoes a reaction cascade analogous to the oxidative dimerization of phenalenyl to peropyrene. By controlling the amount of oxidant used, two intermediates and one side product could be isolated and fully characterized, including single-crystal X-ray diffraction analysis, and two intermediates were detected by electron paramagnetic resonance spectroscopy. In concert with density functional theory calculations, these intermediates support the proposed reaction mechanism. Compared to peropyrene, the absorption and emission of hypercethrene are slightly red-shifted on account of extended Ï-conjugation and the fluorescence quantum yield of 0.45 is decreased by a factor of âŒ2. Enantiomerically enriched hypercethrene displays circularly polarized luminescence with a brightness value of 8.3 M; -1; cm; -1; . Our results show that reactions of graphene-based Ï-radicals-typically considered an "undefined decomposition" of non-zero-spin materials-can be well-defined and selective, and have potential to be transformed into a step-economic synthetic method toward complex carbon nanostructures
Symmetry as a new element to control molecular switches
The isomerization properties of an azocarbazole macrocycle in solution were investigated utilizing NMR spectroscopy with in situ irradiation in combination with DFT calculations. It was demonstrated that the position of azo units in a rigid macrocyclic system influences the photoisomerization pathway even if the initial all-E isomer is highly symmetric. Furthermore, the effect of ring strain on lowering the rates of thermal isomerization was demonstrated and a mechanism via an inversion-rotation proposed. The herein presented results and methods give new insights into the general nature of the azobenzene unit. In particular we illustrate the effect of symmetry changes due to macrocyclic arrangement on the photochemical and thermal isomerization properties, which will stimulate future development towards multinary molecular switches
In-Cell Protein Structures from 2D NMR Experiments
In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples (âŒ50 ÎŒM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures
Structure of formylglycine-generating enzyme in complex with copper and a substrate reveals an acidic pocket for binding and activation of molecular oxygen
The formylglycine generating enzyme (FGE) catalyzes oxidative conversion of specific peptidyl-cysteine residues to formylglycine. FGE mediates O; 2; -activation and hydrogen-atom abstraction in an active site that contains Cu(i) coordinated to two cysteine residues. Similar coordination geometries are common among copper-sensing transcription factors and copper-chaperone but are unprecedented among copper-dependent oxidases. To examine the mechanism of this unusual catalyst we determined the 1.04 Ă
structure of FGE from; Thermomonospora curvata; in complex with copper and a cysteine-containing peptide substrate. This structure unveils a network of four crystallographic waters and two active site residues that form a highly acidic O; 2; -binding pocket juxtaposed to the trigonal planar tris-cysteine coordinated Cu(i) center. Comparison with structures of FGE in complex with Ag(i) and Cd(ii) combined with evidence from NMR spectroscopy and kinetic observations highlight several structural changes that are induced by substrate binding and prime the enzyme for O; 2; -binding and subsequent activation
Electron Transfer across o-Phenylene Wires
Photoinduced electron transfer across rigid rod-like oligo-p-phenylenes has been thoroughly investigated in the past, but their o-connected counterparts are yet entirely unexplored in this regard. We report on three molecular dyads comprised of a triarylamine donor and a Ru(bpy)32+ (bpy =2,2âČ-bipyridine) acceptor connected covalently by 2 to 6 o-phenylene units. Pulsed excitation of the Ru(II) sensitizer at 532 nm leads to the rapid formation of oxidized triarylamine and reduced ruthenium complex via intramolecular electron transfer. The subsequent thermal reverse charge-shift reaction to reinstate the electronic ground-state occurs on a time scale of 120â220 ns in deaerated CH3CN at 25 °C. The conformational flexibility of the o-phenylene bridges causes multiexponential transient absorption kinetics for the photoinduced forward process, but the thermal reverse reaction produces single-exponential transient absorption decays. The key finding is that the flexible o-phenylene bridges permit rapid formation of photoproducts storing ca. 1.7 eV of energy with lifetimes on the order of hundreds of nanoseconds, similar to what is possible with rigid rod-like donorâacceptor compounds. Thus, the conformational flexibility of the o-phenylenes represents no disadvantage with regard to the photoproduct lifetimes, and this is relevant in the greater context of light-to-chemical energy conversion
An Ortho-Tetraphenylene-Based âGelĂ€nderâ Architecture Consisting Exclusively of 52 sp-Hybridized C Atoms
A new type of âGelĂ€nderâ molecule based on a ortho-tetraphenylene core is presented. The central para-quaterphenyl backbone is wrapped by a 4,4â-di((Z)-styryl)-1,1â-biphenyl banister, with its aryl rings covalently attached to all four phenyl rings of the backbone. The resulting helical chiral bicyclic architecture consists exclusively of sp2-hybridized carbon atoms. The target structure was assembled by expanding the central ortho-tetraphenylene subunit with the required additional phenyl rings followed by a twofold macrocyclization. The first macrocyclization attempts based on a twofold McMurry coupling were successful but low yielding; the second strategy, profiting from olefin metathesis, provided satisfying yields. Hydrogenation of the olefins resulted in a saturated derivative of similar topology, thereby allowing the interdependence between saturation and physico-chemical properties to be studied. The target structures, including their solid-state structures, were fully characterized. The helical chiral bicycle was synthesized as a racemate and separated into pure enantiomers by HPLC on a chiral stationary phase. Comparison of recorded and simulated chiroptical properties allowed the enantiomers to be assigned
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