17 O NMR Studies of Yeast Ubiquitin in Aqueous Solution and in the Solid State.
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.
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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
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
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
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
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
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8,219 citations
TL;DR: The crystal structure of human erythrocytic ubiquitin has been refined at 1.8 A resolution using a restrained least-squares procedure and 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.
1,642 citations
TL;DR: 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 as discussed by the authors.
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
1,196 citations
TL;DR: The present chapter focuses primarily on the methodological advances in this latter application, particularly as they relate to the study of proteins in solution.
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.
773 citations
TL;DR: 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, as well as new nuclear magnetic resonance (NMR) experiments developed.
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.
459 citations