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Site‐Specific Isotope‐Labeling of Inosine Phosphoramidites and NMR Analysis of an Inosine‐Containing RNA Duplex
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Dr. Andre Dallmann Dr. Alexander V. Beribisky Dr. Felix Gnerlich Martin Rübbelke Dr. Stefan Schiesser Prof. Dr. Thomas Carell Prof. Dr. Michael Sattler 《Chemistry (Weinheim an der Bergstrasse, Germany)》2016,22(43):15350-15359
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Dr. Hendrik R. A. Jonker Dr. Krishna Saxena Dr. Aleksandra Shcherbakova Birgit Tiemann Prof. Dr. Hans Bakker Prof. Dr. Harald Schwalbe 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(46):20840-20846
Despite the great interest in glycoproteins, structural information reporting on conformation and dynamics of the sugar moieties are limited. We present a new biochemical method to express proteins with glycans that are selectively labeled with NMR-active nuclei. We report on the incorporation of 13C-labeled mannose in the C-mannosylated UNC-5 thrombospondin repeat. The conformational landscape of the C-mannose sugar puckers attached to tryptophan residues of UNC-5 is characterized by interconversion between the canonical 1C4 state and the B03 / 1S3 state. This flexibility may be essential for protein folding and stabilization. We foresee that this versatile tool to produce proteins with selectively labeled C-mannose can be applied and adjusted to other systems and modifications and potentially paves a way to advance glycoprotein research by unravelling the dynamical and conformational properties of glycan structures and their interactions. 相似文献
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Yin Yang Qing‐Feng Li Chan Cao Feng Huang Prof. Xun‐Cheng Su 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(3):1097-1103
Site‐specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biology. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure determination and mobility studies of proteins and protein–ligand complexes. Herein, we present a new way to label proteins in a site‐specific manner with a high‐affinity and chemically stable tag, 4‐vinyl(pyridine‐2,6‐diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: 1) it has a very high binding affinity for lanthanides (higher than EDTA), 2) there is no heterogeneity in complexes with lanthanides, 3) the derivatized protein is stable and potentially applicable to the in situ analysis of proteins. 相似文献
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Untangling a Repetitive Amyloid Sequence: Correlating Biofilm‐Derived and Segmentally Labeled Curli Fimbriae by Solid‐State NMR Spectroscopy
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Dr. Tobias Schubeis Puwei Yuan Dr. Mumdooh Ahmed Dr. Madhu Nagaraj Dr. Barth‐Jan van Rossum Dr. Christiane Ritter 《Angewandte Chemie (International ed. in English)》2015,54(49):14669-14672
Curli are functional bacterial amyloids produced by an intricate biogenesis machinery. Insights into their folding and regulation can advance our understanding of amyloidogenesis. However, gaining detailed structural information of amyloids, and their tendency for structural polymorphisms, remains challenging. Herein we compare high‐quality solid‐state NMR spectra from biofilm‐derived and recombinantly produced curli and provide evidence that they adopt a similar, well‐defined β‐solenoid arrangement. Curli subunits consist of five sequence repeats, resulting in severe spectral overlap. Using segmental isotope labeling, we obtained the unambiguous sequence‐specific resonance assignments and secondary structure of one repeat, and demonstrate that all repeats are most likely structurally equivalent. 相似文献
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Dr. Mariusz Jaremko Dr. Łukasz Jaremko Dr. Saskia Villinger Christian D. Schmidt Prof. Dr. Christian Griesinger Dr. Stefan Becker Prof. Dr. Markus Zweckstetter 《Angewandte Chemie (International ed. in English)》2016,55(35):10518-10521
15N spin‐relaxation rates are demonstrated to provide critical information about the long‐range structure and internal motions of membrane proteins. Combined with an improved calculation method, the relaxation‐rate‐derived structure of the 283‐residue human voltage‐dependent anion channel revealed an anisotropically shaped barrel with a rigidly attached N‐terminal helix. Our study thus establishes an NMR spectroscopic approach to determine the structure and dynamics of mammalian membrane proteins at high accuracy and resolution. 相似文献
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Lukas Sušac Dr. Casey O'Connor Prof. Dr. Raymond C. Stevens Prof. Dr. Kurt Wüthrich 《Angewandte Chemie (International ed. in English)》2015,54(50):15246-15249
We present in‐membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G‐protein‐coupled receptors (GPCRs) with minimal mutagenesis. This method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site‐specific introduction of chromophores for NMR and fluorescence spectroscopy in the human κ‐opioid receptor (KOR) and the human A2A adenosine receptor (A2AAR). IMCM is applicable to a wide range of in vitro studies of GPCRs, including single‐molecule spectroscopy, and is a promising platform for in‐cell spectroscopy experiments. 相似文献
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Nicholle G. A. Bell Adam A. L. Michalchuk John W. T. Blackburn Dr. Margaret C. Graham Dr. Dušan Uhrín 《Angewandte Chemie (International ed. in English)》2015,54(29):8382-8385
Humic substances, the main component of soil organic matter, could form an integral part of green and sustainable solutions to the soil fertility problem. However, their global‐scale application is hindered from both scientific and regulatory perspectives by the lack of understanding of the molecular make‐up of these chromatographically inseparable mixtures containing thousands of molecules. Here we show how multidimensional NMR spectroscopy of isotopically tagged molecules enables structure characterization of humic compounds. We illustrate this approach by identifying major substitution patterns of phenolic aromatic moieties of a peat soil fulvic acid, an operational fraction of humic substances. Our methodology represents a paradigm shift in the use of NMR active tags in structure determination of small molecules in complex mixtures. Unlike previous tagging methodologies that focused on the signals of the tags, we utilize tags to directly probe the identity of the molecules they are attached to. 相似文献
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Functional Dynamics of Deuterated β2‐Adrenergic Receptor in Lipid Bilayers Revealed by NMR Spectroscopy
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Dr. Yutaka Kofuku Dr. Takumi Ueda Junya Okude Yutaro Shiraishi Keita Kondo Takuya Mizumura Shiho Suzuki Prof. Dr. Ichio Shimada 《Angewandte Chemie (International ed. in English)》2014,53(49):13376-13379
G‐protein‐coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β2‐adrenergic receptor (β2AR) in lipid bilayers of reconstituted high‐density lipoprotein (rHDL) increased by approximately 5‐fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β2AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception. 相似文献
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Dr. Brent G. Pautler Christopher A. Colla Dr. Rene L. Johnson Peter Klavins Dr. Stephen J. Harley Dr. C. André Ohlin Prof. Dimitri A. Sverjensky Dr. Jeffrey H. Walton Prof. William H. Casey 《Angewandte Chemie (International ed. in English)》2014,53(37):9788-9791
A non‐magnetic piston‐cylinder pressure cell is presented for solution‐state NMR spectroscopy at geochemical pressures. The probe has been calibrated up to 20 kbar using in situ ruby fluorescence and allows for the measurement of pressure dependencies of a wide variety of NMR‐active nuclei with as little as 10 μL of sample in a microcoil. Initial 11B NMR spectroscopy of the H3BO3–catechol equilibria reveals a large pressure‐driven exchange rate and a negative pressure‐dependent activation volume, reflecting increased solvation and electrostriction upon boron‐catecholate formation. The inexpensive probe design doubles the current pressure range available for solution NMR spectroscopy and is particularly important to advance the field of aqueous geochemistry. 相似文献