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ABSTRACT: Site-specific attachment of paramagnetic lanthanide ions to a protein generates pseudocontact shifts (PCS) in the nuclear magnetic resonance (NMR) spectra of the protein that are easily measured as changes in chemical shifts. By labeling the protein with lanthanide tags at four different sites, PCSs are observed for most amide protons and accurate information is obtained about their coordinates in three-dimensional space. The approach is demonstrated with the chaperone ERp29, for which large differences have been reported between X-ray and NMR structures of the C-terminal domain, ERp29-C. The results unambiguously show that the structure of rat ERp29-C in solution is similar to the crystal structure of human ERp29-C. PCSs of backbone amides were the only structural restraints required. Because these can be measured for more dilute protein solutions than other NMR restraints, the approach greatly widens the range of proteins amenable to structural studies in solution.
Structure 04/2013; · 6.35 Impact Factor
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Kiyoshi Ozawa,
Nicholas P Horan,
Andrew Robinson,
Hiromasa Yagi,
Flynn R Hill,
Slobodan Jergic,
Zhi-Qiang Xu,
Karin V Loscha,
Nan Li,
Moeava Tehei,
Aaron J Oakley,
Gottfried Otting, Thomas Huber,
Nicholas E Dixon
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ABSTRACT: A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β2 replicase complex with primer-template DNA combine all available structural data.
Nucleic Acids Research 04/2013; · 8.03 Impact Factor
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Slobodan Jergic,
Nicholas P Horan,
Mohamed M Elshenawy,
Claire E Mason,
Thitima Urathamakul,
Kiyoshi Ozawa,
Andrew Robinson,
Joris M H Goudsmits,
Yao Wang,
Xuefeng Pan,
Jennifer L Beck,
Antoine M van Oijen, Thomas Huber,
Samir M Hamdan,
Nicholas E Dixon
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ABSTRACT: Processive DNA synthesis by the αɛθ core of the Escherichia coli Pol III replicase requires it to be bound to the β(2) clamp via a site in the α polymerase subunit. How the ɛ proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ɛ. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in ɛ that, in conjunction with the site in α, maintains a closed state of the αɛθ-β(2) replicase in the polymerization mode of DNA synthesis. The ɛ-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.
The EMBO Journal 02/2013; · 9.20 Impact Factor
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ABSTRACT: Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide-alkyne cycloaddition to a genetically encoded p-azido-L-phenylalanine residue. The resulting tether proved sufficiently rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.
Bioconjugate Chemistry 01/2013; · 4.93 Impact Factor
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ABSTRACT: Double electron-electron resonance (DEER) at W-band (95GHz) was applied to measure the distance between a pair of nitroxide and Gd(3+) chelate spin labels, about 6nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700MHz) between the narrow, intense signal of the Gd(3+) central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12μs in a protein. This opens the possibility of accessing distances of 8nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd(3+)-nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels.
Journal of Magnetic Resonance 12/2012; 227C:66-71. · 2.14 Impact Factor
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ABSTRACT: The pulse DEER (Double Electron-Electron Resonance) technique is frequently applied for measuring nanometer distances between specific sites in biological macromolecules. In this work we extend the applicability of this method to high field distance measurements in a protein assembly with mixed spin labels, i.e. a nitroxide spin label and a Gd(3+) tag. We demonstrate the possibility of spectroscopic selection of distance distributions between two nitroxide spin labels, a nitroxide spin label and a Gd(3+) ion, and two Gd(3+) ions. Gd(3+)-nitroxide DEER measurements possess high potential for W-band long range distance measurements (6 nm) by combining high sensitivity with ease of data analysis, subject to some instrumental improvements.
Physical Chemistry Chemical Physics 04/2012; 14(13):4355-8. · 3.57 Impact Factor
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ABSTRACT: Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Δχ-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Δχ-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge of the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations.
Journal of Molecular Biology 03/2012; 416(5):668-77. · 4.00 Impact Factor
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ABSTRACT: A cell-free protein synthesis system from which the release factor RF1 has been selectively removed enables the facile incorporation of unnatural amino acids into proteins at difficult and multiple sites by optimized use of orthogonal tRNA/aminoacyl-tRNA synthetase systems. (19) F NMR spectroscopy of a protein labeled combinatorially with trifluoromethyl phenylalanine (red in picture) at multiple sites establishes resonance assignments with a minimal number of samples.
Angewandte Chemie International Edition 02/2012; 51(9):2243-6. · 13.45 Impact Factor
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ABSTRACT: Pulse electron paramagnetic resonance measurements of long-range (nm scale) distances between spin labels site-specifically attached to biomacromolecules have proven highly effective in structural studies. The most commonly used spin labels are stable nitroxide radicals, and measurements are usually carried out at X-band frequencies (9.5 GHz, 0.35 T). Higher magnetic fields open new possibilities for distance measurements with increased sensitivity using alternative spin labels containing half-integer high-spin metal ions. Here we demonstrate W-band (95 GHz) pulse double electron–electron resonance (DEER) distance measurements in a protein labeled with two Mn2+-EDTA tags. The distance distribution obtained is in excellent agreement with model calculations based on the known solution NMR structure. Thus, site-specific labeling with Mn2+ tags opens a highly promising approach to nanometer distance measurements in biological macromolecules.Keywords: EPR; DEER; PELDOR; high field; spin labeling; distance measurements; Mn2+-EDTA
01/2012;
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ABSTRACT: The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro ("open state"), necessitating a large structural change to produce the "closed state" thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of (15)N-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd(3+) complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned (15)N-HSQC spectra.
Journal of the American Chemical Society 11/2011; 133(47):19205-15. · 9.91 Impact Factor
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Bim Graham,
Choy Theng Loh,
James David Swarbrick,
Phuc Ung,
James Shin,
Hiromasa Yagi,
Xinying Jia,
Sandeep Chhabra,
Nicholas Barlow,
Guido Pintacuda, Thomas Huber,
Gottfried Otting
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ABSTRACT: Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.
Bioconjugate Chemistry 08/2011; 22(10):2118-25. · 4.93 Impact Factor
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ABSTRACT: Paramagnetic relaxation enhancements from unpaired electrons observed in nuclear magnetic resonance (NMR) spectra present powerful long-range distance restraints. The most frequently used paramagnetic tags, however, are tethered to the protein via disulfide bonds, requiring proteins with single cysteine residues for covalent attachment. Here we present a straightforward strategy to tag proteins site-specifically with paramagnetic lanthanides without a tether and independent of cysteine residues. It relies on preferential binding of the complex between three dipicolinic acid molecules (DPA) and a lanthanide ion (Ln(3+)), [Ln(DPA)(3)](3-), to a pair of positively charged amino acids whose charges are not compensated by negatively charged residues nearby. This situation rarely occurs in wild-type proteins, allowing the creation of specific binding sites simply by introduction of positively charged residues that are positioned far from glutamate or aspartate residues. The concept is demonstrated with the hnRNPLL RRM1 domain. In addition, we show that histidine- and arginine-tags present binding sites for [Ln(DPA)(3)](3-).
Journal of Biomolecular NMR 08/2011; 50(4):411-20. · 3.61 Impact Factor
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ABSTRACT: Attachment of two nitrilotriacetic acid-based ligands to a protein α-helix in an i, i + 4 configuration produces an octadentate chelating motif that is able to bind paramagnetic lanthanide ions rigidly and with high affinity, leading to large pseudocontact shifts and residual dipolar couplings in the NMR spectrum.
Chemical Communications 07/2011; 47(26):7368-70. · 6.17 Impact Factor
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ABSTRACT: Double electron-electron resonance (DEER) distance measurements of a protein complex tagged with two Gd(3+) chelates developed for rigid positioning of the metal ion are shown to deliver outstandingly accurate distance measurements in the 6 nm range. The accuracy was assessed by comparison with modeled distance distributions based on the three-dimensional molecular structures of the protein and the tag and further comparison with paramagnetic NMR data. The close agreement between the predicted and experimentally measured distances opens new possibilities for investigating the structure of biomolecular assemblies. As an example, we show that the dimer interface of rat ERp29 in solution is the same as that determined previously for human ERp29 in the single crystal.
Journal of the American Chemical Society 06/2011; 133(27):10418-21. · 9.91 Impact Factor
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ABSTRACT: Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structure restraints for studies of proteins by nuclear magnetic resonance spectroscopy. To elicit PCSs, the lanthanide must be attached site-specifically to the target protein. In addition, it needs to be attached rigidly to avoid averaging of the PCSs due to mobility with respect to the protein and it must not interfere with the function of the protein. Here, we present a dipicolinic acid reagent that spontaneously forms a disulfide bond with thiol groups of accessible cysteine residues. A minimal number of rotatable bonds between the cysteine side chain and the tag helps to minimise mobility. Combined with the small size of the tag and quantitative tagging yields, these features make it a highly attractive tool for generating structure restraints by paramagnetic lanthanides.
Chemistry 06/2011; 17(24):6830-6. · 5.93 Impact Factor
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Angewandte Chemie International Edition 01/2011; 50(3):692-4. · 13.45 Impact Factor
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09/2010: pages 265 - 277; , ISBN: 9780470882207
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ABSTRACT: Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd(3+) spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and tau(C)14, labeled at strategically selected sites with either two nitroxides or two Gd(3+) spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd(3+) tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (approximately 9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd(3+)-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd(3+)-Gd(3+) distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein tau(C)14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd(3+)-Gd(3+) distance distribution with a maximum at 3.4 nm. The Gd(3+)-Gd(3+) distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd(3+) labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection.
Journal of the American Chemical Society 07/2010; 132(26):9040-8. · 9.91 Impact Factor
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ABSTRACT: Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein-ligand complexes. In contrast to conventional PRE reagents that are covalently attached to the protein, the complex between gadolinium and three dipicolinic acid (DPA) molecules, [Gd(DPA)(3)](3-), can bind to proteins in a non-covalent yet site-specific manner. This offers straightforward access to PREs that can be scaled by using different ratios of [Gd(DPA)(3)](3-) to protein, allowing quantitative distance measurements for nuclear spins within about 15 A of the Gd(3+) ion. Such data accurately define the metal position relative to the protein, greatly enhancing the interpretation of pseudocontact shifts induced by [Ln(DPA)(3)](3-) complexes of paramagnetic lanthanide (Ln(3+)) ions other than gadolinium. As an example we studied the quaternary structure of the homodimeric GCN4 leucine zipper.
Journal of Biomolecular NMR 06/2010; 47(2):143-53. · 3.61 Impact Factor
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ABSTRACT: Paramagnetic effects from lanthanide ions present powerful tools for protein studies by nuclear magnetic resonance (NMR) spectroscopy provided that the lanthanide can be site-specifically and rigidly attached to the protein. A new, particularly small and rigid lanthanide-binding tag, 3-mercapto-2,6-pyridinedicarboxylic acid (3MDPA), was synthesized and attached to two different proteins via a disulfide bond. The complexes of the N-terminal domain of the E. coli arginine repressor (ArgN) with seven different paramagnetic lanthanide ions and Co(2+) were analyzed in detail by NMR spectroscopy. The magnetic susceptibility anisotropy (Delta chi) tensors and metal position were determined from pseudocontact shifts. The 3MDPA tag generated very different Delta chi tensor orientations compared to the previously studied 4-mercaptomethyl-DPA tag, making it a highly complementary and useful tool for protein NMR studies.
Chemistry 02/2010; 16(12):3827-32. · 5.93 Impact Factor
