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Open accessJournal ArticleDOI: 10.1002/CBIC.202000659

17 O NMR Studies of Yeast Ubiquitin in Aqueous Solution and in the Solid State.

02 Mar 2021-ChemBioChem (John Wiley & Sons, Ltd)-Vol. 22, Iss: 5, pp 826-829
Abstract: We report a general method for amino acid-type specific 17 O-labeling of recombinant proteins in Escherichia coli. In particular, we have prepared several [1-13 C,17 O]-labeled yeast ubiquitin (Ub) samples including Ub-[1-13 C,17 O]Gly, Ub-[1-13 C,17 O]Tyr, and Ub-[1-13 C,17 O]Phe using the auxotrophic E. coli strain DL39 GlyA λDE3 (aspC- tyrB- ilvE- glyA- λDE3). We have also produced Ub-[η-17 O]Tyr, in which the phenolic group of Tyr59 is 17 O-labeled. We show for the first time that 17 O NMR signals from protein terminal residues and side chains can be readily detected in aqueous solution. We also reported solid-state 17 O NMR spectra for Ub-[1-13 C,17 O]Tyr and Ub-[1-13 C,17 O]Phe obtained at an ultrahigh magnetic field, 35.2 T (1.5 GHz for 1 H). This work represents a significant advance in the field of 17 O NMR studies of proteins.

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6 results found


Journal ArticleDOI: 10.1148/RADIOLOGY.204.1.100
01 Jul 1997-Radiology
Abstract: The pleckstrin DEP domain structure DEP domains are divergent in sequence. Using multiple sequence alignments and phylogenetic tree reconstructions, we found at least six subfamilies of DEP domains. We selected Pleckstrin, the major substrate of PKC in platelets, for further structural research. We found that the Pleck-DEP fold contains an additional short helix !4 inserted in the "4-"5 loop with respect to other DEP structures. This helix exhibits increased backbone mobility, as shown by NMR relaxation measurements, and may be involved in protein-protein interactions. (Civera et al, 2005.)

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40 Citations


Journal ArticleDOI: 10.1039/D1NJ02847B
Abstract: We report the solid-state 17O NMR parameters for five previously uncharacterized N-α-fluoren-9-yl-methoxycarbonyl-O-t-butyl (Fmoc) protected amino acids. These molecules are critical to constructing synthetic biological systems, like peptides, and provide an avenue for introducing 17O as an NMR probe nucleus. A multiple-turnover reaction was used to efficiently 17O label the carboxylic acid moieties of Fmoc-L-isoleucine, Fmoc-L-tryptophan, Fmoc-L-proline, Fmoc-L-tyrosine and Fmoc-L-threonine. Magic-angle spinning (MAS) and non-spinning NMR spectra were obtained at two magnetic field strengths (14.1 and 21.1 T) and the quadrupolar and chemical shift parameters for the carbonyl and hydroxyl sites were determined. Computed NMR parameters using density functional theory (DFT) were found to be in good agreement with experimental results, supporting the identification of minor unprotonated species present. This work continues to highlight 17O as a sensitive probe nucleus of its local environment and reinforces the importance of developing solid-state 17O NMR techniques to expand the analytical NMR toolkit for exploring biologically relevant molecules.

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Topics: NMR spectra database (52%), Carboxylic acid (51%)

1 Citations


Journal ArticleDOI: 10.1021/ACS.JPCLETT.1C02465
Ivan Hung, Zhehong Gan, Gang Wu1Institutions (1)
Abstract: We report two- and three-dimensional (2D and 3D) 13C-17O heteronuclear correlation solid-state NMR experiments under magic-angle spinning (MAS) conditions. These experiments utilize the D-RINEPT (Dipolar-mediated Refocused Insensitive Nuclei Enhanced by Polarization Transfer) scheme with symmetry-based SR412 recoupling blocks for coherence transfer between 13C and 17O nuclei. First, a 2D 17O → 13C correlation experiment was performed for the [1-13C,17O]-Gly/Gly·HCl cocrystal and [U-13C, 1-17O]-α/β-d-glucose samples. Second, a 2D 17O → 13C MQ-D-RINEPT correlation experiment where the indirect dimension incorporates the multiple-quantum MAS (MQMAS) scheme was tested for obtaining isotropic 17O resolution with [U-13C, 1-17O]-α/β-d-glucose. Third, a new 3D 17O → 13C → 13C correlation experiment was demonstrated where 17O → 13C and 13C → 13C correlations are achieved by D-RINEPT and DARR (Dipolar Assisted Rotational Resonance) sequences, respectively (thus termed as a 3D D-RINEPT/DARR OCC experiment). This new 3D 17O NMR experiment is implemented with the aim for site-resolved solid-state 17O NMR studies.

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1 Citations


Book ChapterDOI: 10.1016/BS.ARNMR.2021.03.001
James Palmer1, Gang Wu1Institutions (1)
01 Jan 2021-
Abstract: This chapter reviews recent advances (published since 2014 in the literature) in the field of solid-state 17O NMR studies of organic and biological molecules. New results are grouped into the following categories: (1) organic compounds, (2) metal organic frameworks, (3) hydrogen-bonded systems, (4) paramagnetic coordination compounds, (5) peptides and proteins, and (6) dynamic nuclear polarization.

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Topics: Coordination complex (51%)

1 Citations


Open accessJournal ArticleDOI: 10.3390/BIOLOGY10060453
21 May 2021-Biology
Abstract: Oxygen is a key atom that maintains biomolecular structures, regulates various physiological processes, and mediates various biomolecular interactions. Oxygen-17 (17O), therefore, has been proposed as a useful probe that can provide detailed information about various physicochemical features of proteins. This is attributed to the facts that (1) 17O is an active isotope for nuclear magnetic resonance (NMR) spectroscopic approaches; (2) NMR spectroscopy is one of the most suitable tools for characterizing the structural and dynamical features of biomolecules under native-like conditions; and (3) oxygen atoms are frequently involved in essential hydrogen bonds for the structural and functional integrity of proteins or related biomolecules. Although 17O NMR spectroscopic investigations of biomolecules have been considerably hampered due to low natural abundance and the quadruple characteristics of the 17O nucleus, recent theoretical and technical developments have revolutionized this methodology to be optimally poised as a unique and widely applicable tool for determining protein structure and dynamics. In this review, we recapitulate recent developments in 17O NMR spectroscopy to characterize protein structure and folding. In addition, we discuss the highly promising advantages of this methodology over other techniques and explain why further technical and experimental advancements are highly desired.

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52 results found


Open accessJournal ArticleDOI: 10.1051/EPN/19861701011
01 Jan 1986-Europhysics News
Topics: Nucleic acid (60%)

8,146 Citations


Journal ArticleDOI: 10.1016/0022-2836(87)90679-6
Abstract: The crystal structure of human erythrocytic ubiquitin has been refined at 1.8 A resolution using a restrained least-squares procedure. The crystallographic R-factor for the final model is 0.176. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.5 degrees, respectively. A total of 58 water molecules per molecule of ubiquitin are included in the final model. The last four residues in the molecule appear to have partial occupancy or large thermal motion. The overall structure of ubiquitin is extremely compact and tightly hydrogen-bonded; approximately 87% of the polypeptide chain is involved in hydrogen-bonded secondary structure. Prominent secondary structural features include three and one-half turns of alpha-helix, a short piece of 3(10)-helix, a mixed beta-sheet that contains five strands, and seven reverse turns. There is a marked hydrophobic core formed between the beta-sheet and alpha-helix. The molecule features a number of unusual secondary structural features, including a parallel G1 beta-bulge, two reverse Asx turns, and a symmetrical hydrogen-bonding region that involves the two helices and two of the reverse turns.

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1,551 Citations


Journal ArticleDOI: 10.1021/JA00124A023
Abstract: Interest in the solid state nuclear magnetic resonance (NMR) spectroscopy of half-integer quadrupolar spins is strongly stimulated by the roles that these isotopes play in a variety of important systems such as minerals, structural ceramics, semiconductors, glasses, and catalysts.' In spite of the partly ionic nature of these materials, quadrupole interactions with surrounding electric field gradients often broaden the solid state NMR line shapes of these nuclei into the MHz range. Although most of this broadening can be circumvented by limiting excitations to the central -l/2 +l/2 transition? second-order effects can widen the resulting resonances and prevent the resolution of chemically inequivalent sites. In contrast to what happens in spin-'/2 spectroscopy, no single-axis spinning techniques are available for canceling the effects of these second-order anisotropies. Still, as is briefly discussed in the present Communication, bidimensional NMR methods involving multiple-quantum excitation in combination with fixed-angle sample spinning are capable of refocusing second-order quadrupolar effects and can thus be used to acquire highly resolved spectra devoid from quadrupolar, shielding, or dipolar anisotropies. Central-transition NMR experiments manage to avoid firstorder quadrupolar broadenings owing to the Hamiltonian's quadratic dependence on the S, angular momentum.2 The following term in the quadrupolee the last two, however, are anisotropic and can broaden the central transitions of powdered samples over several kHz. The effects of these anisotropies can be scaled by rapidly spinning the sample at an axis Ps3 This leads to time averaged NMR frequencies

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Topics: Magic angle spinning (60%), Solid-state nuclear magnetic resonance (58%), Magic angle (56%) ... read more

1,150 Citations


Book ChapterDOI: 10.1007/978-1-349-12749-8_2
Abstract: Since the first experimental observation of nuclear magnetic resonance (NMR) in bulk matter more than 45 years ago (Bloch et al., 1946; Purcell et al., 1946), its history has been punctuated by a series of revolutionary advances that have greatly expanded its horizons. Indeed, methodological and instrumental developments witnessed over the past two decades have turned NMR into the most diverse spectroscopic tool currently available. Applications vary from exploration of natural resources and medical imaging to determination of the three-dimensional structure of biologically important macromolecules (Wuthrich, 1986; Kaptein et al., 1988; Bax, 1989; Clore and Gronenborn, 1989; Markley, 1989; Wuthrich, 1989; Wagner et al., 1992). The present chapter focuses primarily on the methodological advances in this latter application, particularly as they relate to the study of proteins in solution.

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772 Citations


Book ChapterDOI: 10.1016/0076-6879(89)77005-1
Abstract: Publisher Summary This chapter discusses the methods of biosynthetic enrichment of proteins with 15N in backbone and side-chain groups, as exemplified by the work with bacteriophage T4 lysozyme. Three necessary features for the investigation of 15N-labeled proteins are discussed. First, the protein must be rapidly and efficiently produced in milligram quantities. Second, the protein must be uniformly or selectively enriched in 15N by growth of the bacteria on defined media supplemented with the isotope. Third, new nuclear magnetic resonance (NMR) experiments have been developed, which either directly or indirectly yield information regarding the 15N nucleus or exploit the 15N as a means to filter 1H NMR spectra. One of the goals of the research on T4 lysozyme is to investigate the effect of amino acid substitutions on the structure and dynamics of the protein. Unfortunately, in three cases, it has been observed that many changes in the 15N- 1H spectra of uniformly 15N-labeled mutant lysozymes relative to the wild-type protein. This limits the extension of assignments of resonances from the wild-type to mutant lysozymes.

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Topics: Protein structure (56%), Nuclear magnetic resonance spectroscopy (56%), Proton NMR (52%) ... read more

454 Citations