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Ivan Hung

Bio: Ivan Hung is an academic researcher. The author has an hindex of 1, co-authored 1 publications receiving 6 citations.

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TL;DR: It is shown for the first time that 17O NMR signals from protein terminal residues and side chains can be readily detected in aqueous solution and represents a significant advance in the field of 17ONMR studies of proteins.
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.

15 citations


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TL;DR: It is found that the Pleck-DEP fold contains an additional short helix !
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.)

41 citations

Journal ArticleDOI
TL;DR: In this paper , the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design, was investigated.
Abstract: Abstract Aggregation of amyloidogenic proteins is a characteristic of multiple neurodegenerative diseases. Atomic resolution of small molecule binding to such pathological protein aggregates is of interest for the development of therapeutics and diagnostics. Here we investigate the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design. We used nuclear magnetic resonance spectroscopy and the cryogenic electron microscopy structure of α-synuclein fibrils grown in the presence of lipids to locate anle138b within a cavity formed between two β-strands. We explored and quantified multiple binding modes of the compound in detail using molecular dynamics simulations. Our results reveal stable polar interactions between anle138b and backbone moieties inside the tubular cavity of the fibrils. Such cavities are common in other fibril structures as well.

12 citations

Journal ArticleDOI
TL;DR: In this paper , the authors report synthesis and solid-state 17O NMR characterization of α-d-glucose for which all six oxygen atoms are site-specifically 17O-labeled.
Abstract: We report synthesis and solid-state 17O NMR characterization of α-d-glucose for which all six oxygen atoms are site-specifically 17O-labeled. Solid-state 17O NMR spectra were recorded for α-d-glucose/NaCl/H2O (2/1/1) cocrystals under static and magic-angle-spinning (MAS) conditions at five moderate, high, and ultrahigh magnetic fields: 14.1, 16.4, 18.8, 21.1, and 35.2 T. Complete 17O chemical shift (CS) and quadrupolar coupling (QC) tensors were determined for each of the six oxygen-containing functional groups in α-d-glucose. Paramagnetic Cu(ii) doping was found to significantly shorten the spin–lattice relaxation times for both 1H and 17O nuclei in these compounds. A combination of the paramagnetic Cu(ii) doping, new CPMAS CryoProbe technology, and apodization weighted sampling led to a sensitivity boost for solid-state 17O NMR by a factor of 6–8, which made it possible to acquire high-quality 2D 17O multiple-quantum (MQ) MAS spectra for carbohydrate compounds. The unprecedented spectral resolution offered by 2D 17O MQMAS spectra permitted detection of a key structural difference for a single hydrogen bond between two types of crystallographically distinct α-d-glucose molecules. This work represents the first case where all oxygen-containing functional groups in a carbohydrate molecule are site-specifically 17O-labeled and fully characterized by solid-state 17O NMR. Gauge Including Projector Augmented Waves (GIPAW) DFT calculations were performed to aid 17O and 13C NMR signal assignments for a complex crystal structure where there are six crystallographically distinct α-d-glucose molecules in the asymmetric unit.

10 citations

Journal ArticleDOI
TL;DR: In this paper, an improved multiple-turnover labeling procedure was proposed to develop a fast and cost-effective method to label fluoroenylmethyloxycarbonyl (Fmoc)-protected amino acid building blocks.
Abstract: Solid-state 1H, 13C, and 15N nuclear magnetic resonance (NMR) spectroscopy has been an essential analytical method in studying complex molecules and biomolecules for decades. While oxygen-17 (17O) NMR is an ideal and robust candidate to study hydrogen bonding within secondary and tertiary protein structures for example, it continues to elude many. We discuss an improved multiple-turnover labeling procedure to develop a fast and cost-effective method to 17O label fluoroenylmethyloxycarbonyl (Fmoc)-protected amino acid building blocks. This approach allows for inexpensive ($0.25 USD/mg) insertion of 17O labels, an important barrier to overcome for future biomolecular studies. The 17O NMR results of these building blocks and a site-specific strategy for labeled N-acetyl-MLF-OH and N-formyl-MLF-OH tripeptides are presented. We showcase growth in NMR development for maximizing sensitivity gains using emerging sensitivity enhancement techniques including population transfer, high-field dynamic nuclear polarization, and cross-polarization magic-angle spinning cryoprobes.

6 citations

Journal ArticleDOI
TL;DR: In this paper , the use of 17O for initial polarization is found to provide better sensitivity per unit time compared to 1H, and the potential to determine sequential assignments and long-range distance restraints is demonstrated by using 3D 1H/13C/17O experiments, suggesting that such methods can become an essential tool for biomolecular structure determination.
Abstract: Oxygen is an integral component of proteins but remains sparsely studied because its only NMR active isotope, 17O, has low sensitivity, low resolution, and large quadrupolar couplings. These issues are addressed here with efficient isotopic labeling, high magnetic fields, fast sample spinning, and 1H detection in conjunction with multidimensional experiments to observe oxygen sites specific to each amino acid residue. Notably, cross-polarization at high sample spinning frequencies provides efficient 13C ↔ 17O polarization transfer. The use of 17O for initial polarization is found to provide better sensitivity per unit time compared to 1H. Sharp isotropic 17O peaks are obtained by using a low-power multiple-quantum sequence, which in turn allows extraction of quadrupolar parameters for each oxygen site. Finally, the potential to determine sequential assignments and long-range distance restraints is demonstrated by using 3D 1H/13C/17O experiments, suggesting that such methods can become an essential tool for biomolecular structure determination.

6 citations