Efficient χ-tensor determination and NH assignment of paramagnetic proteins

Anisotropic magnetic susceptibility tensors χ of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the χ-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D ¹⁵N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable χ-tensor anisotropy parameters from 2D spectra of uniformly ¹⁵N-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit ε from E. coli DNA polymerase III in complex with the subunit θ and La³⁺ in its diamagnetic and Dy³⁺, Tb³⁺, and Er³⁺ in its paramagnetic form.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.The first two authors contributed equally to the project.     

Positive φ-angles in proteins by nuclear magnetic resonance spectroscopy

Summary Non-glycine residues with positive -angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) ofBacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant and integration of the nuclear Overhauser H^N-H cross peak, positive -angles could be determined reliably to 60°±30°, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2. The work emphasizes that positive -angles can also occur in non-glycine residues and in the four proteins, positive -angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured coupling constants and the intensity of the intraresidue H^N-H NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wüthrich, K. (1984)J. Mol. Biol.,180, 741–751; Ludvigsen, S., Andersen, K.V. and Poulsen, F.M. (1991)J. Mol. Biol.,217, 731–736).     

Recognition of nuclear targeting signals by Karyopherin-β proteins

The Karyopherin-β family of nuclear transport factors mediates the majority of nucleocytoplasmic transport. Although each of the 19 Karyopherin-βs transports unique sets of cargos, only three classes of nuclear localization and export signals, or NLSs and NESs, have been characterized. The short basic classical-NLS was first discovered in the 1980s and their karyopherin-bound structures were first reported more than 10 years ago. More recently, structural and biophysical studies of Karyopherin-β2-cargo complexes led to definition of the complex and diverse PY-NLS. Structural knowledge of the leucine-rich NES is finally available more than 10 years after the discovery of its recognition by the exportin CRM1. We review recent findings relating to how these three classes of nuclear targeting signals are recognized by their Karyopherin-β nuclear transport factors.     

Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13Cα nuclear spin relaxation

Current methods of determining the rotational diffusion tensors of proteins in solution byNMR spectroscopy exclusively utilize relaxation rate constants for backbone amide 15N spins.However, the distributions of orientations of N-H bond vectors are not isotropic in manyproteins, and correlations between bond vector orientations reduce the accuracy and precisionof rotational diffusion tensors extracted from 15N spin relaxation data. The inclusion of both13C and 15N spin relaxation rate constants increases the robustness of the diffusiontensor analysis because the orientations of the C-H bond vectors differ from theorientations of the N-H bond vectors. Theoretical and experimental results for calbindin D9k,granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributionsof N-H and C-H bond vectors, are used to illustrate the advantages of thesimultaneous utilization of 13C and 15N relaxation data.     

NMR relaxation parameters of methyl groups as a tool to map the interfaces of helix–helix interactions in membrane proteins

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation.     

Complete assignment of Ala,Ile, Leu,Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl–methyl NOESY

Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl–methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.     

Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.     

Measurement of ¹⁵N relaxation rates in perdeuterated proteins by TROSY-based methods

While extracting dynamics parameters from backbone (15)N relaxation measurements in proteins has become routine over the past two decades, it is increasingly recognized that accurate quantitative analysis can remain limited by the potential presence of systematic errors associated with the measurement of (15)N R(1) and R(2) or R(1ρ) relaxation rates as well as heteronuclear (15)N-{(1)H} NOE values. We show that systematic errors in such measurements can be far larger than the statistical error derived from either the observed signal-to-noise ratio, or from the reproducibility of the measurement. Unless special precautions are taken, the problem of systematic errors is shown to be particularly acute in perdeuterated systems, and even more so when TROSY instead of HSQC elements are used to read out the (15)N magnetization through the NMR-sensitive (1)H nucleus. A discussion of the most common sources of systematic errors is presented, as well as TROSY-based pulse schemes that appear free of systematic errors to the level of <1 %. Application to the small perdeuterated protein GB3, which yields exceptionally high S/N and therefore is an ideal test molecule for detection of systematic errors, yields relaxation rates that show considerably less residue by residue variation than previous measurements. Measured R(2)'/R(1)' ratios fit an axially symmetric diffusion tensor with a Pearson's correlation coefficient of 0.97, comparable to fits obtained for backbone amide RDCs to the Saupe matrix.     

Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins?

Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane- bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein- framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes.     

Measurement of rate constants for homodimer subunit exchange using double electron–electron resonance and paramagnetic relaxation enhancements

Here, we report novel methods to measure rate constants for homodimer subunit exchange using double electron–electron resonance (DEER) electron paramagnetic resonance spectroscopy measurements and nuclear magnetic resonance spectroscopy based paramagnetic relaxation enhancement (PRE) measurements. The techniques were demonstrated using the homodimeric protein Dsy0195 from the strictly anaerobic bacterium Desulfitobacterium hafniense Y51. At specific times following mixing site-specific MTSL-labeled Dsy0195 with uniformly ¹⁵N-labeled Dsy0195, the extent of exchange was determined either by monitoring the decrease of MTSL-labeled homodimer from the decay of the DEER modulation depth or by quantifying the increase of MTSL-labeled/¹⁵N-labeled heterodimer using PREs. Repeated measurements at several time points following mixing enabled determination of the homodimer subunit dissociation rate constant, k _?1, which was 0.037 ± 0.005 min^?1 derived from DEER experiments with a corresponding half-life time of 18.7 min. These numbers agreed with independent measurements obtained from PRE experiments. These methods can be broadly applied to protein–protein and protein-DNA complex studies.     

Nuclear overhauser effects for methyl β-maltoside and the conformational states of maltose in aqueous solution

A nuclear Overhauser enhancement in methyl β-maltoside, resulting from pre-irradiation of H-1′ of the non-reducing glucose residue, has been measured and calculated theoretically. Comparison of these data reveals a complicated conformational equilibrium in aqueous solutions of maltose derivatives.     

Hadamard NMR spectroscopy for relaxation measurements of large (>35 kDa) proteins

Here we present a suite of pulse sequences for the measurement of ¹⁵N T₁, T_1ρ and NOE data that combine traditional TROSY-based pulse sequences with band-selective Hadamard frequency encoding. The additive nature of the Hadamard matrix produces much reduced resonance overlap without the need for an increase in the dimensionality of the experiment or a significant decrease in the signal to noise ratio. We validate the accuracy of these sequences in application to ubiquitin and demonstrate their utility for relaxation measurements in Escherichia coli Class II fructose 1,6-bisphosphate aldolase (FBP-aldolase), a 358 residue 78 kDa dimeric enzyme. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The detection and characterization by electron-paramagnetic-resonance spectroscopy of iron–sulphur proteins and other electron-transport components in chromatophores from the purple bacterium Chromatium

Low-temperature e.p.r. (electron-paramagnetic-resonance) spectroscopy was used to detect electron-transport components in Chromatium chromatophores with e.p.r. signals in the g=2.00 region. High-potential iron protein (E(m8.0)=+325mV, where E(m8.0) is the midpoint potential at pH8) and a second component (g=1.90, E(m8.0)=+285mV) are oxidized in illuminated chromatophores. Two iron-sulphur proteins (g=1.94) with E(m8.0)=-290mV and E(m8.0)=-50mV are present. One (E(m8.0)=-50mV) is reduced on illumination. A component (g=1.82) with E(m8.0)=-135mV is photoreduced at 10 degrees K. The midpoint potential of this component is altered by o-phenanthroline and pH. The properties of this component suggest that it is the primary electron acceptor of a photochemical system. Another component (g=1.98) also has some of the properties of a primary electron acceptor, but its function cannot be completely defined. These results show that iron-sulphur proteins are present in the electron-transport system of Chromatium and indicate their role in electron transport.     

Determination of sugar conformation in large RNA oligonucleotides from analysis of dipole–dipole cross correlated relaxation by solution NMR spectroscopy

A new experiment, the forward directed quantitative -HCCH-TOCSY for the measurement of the conformation of the five-membered ribosyl unit in RNA oligonucleotides, is presented. The experiment relies on quantification of cross peak intensities caused by evolution of CH,CH-dipole–dipole cross correlated relaxation in non-evolution periods and the resolution enhancement obtainable in forward directed HCC-TOCSY transfer. Cross correlated relaxation rates are interpreted to reveal the sugar conformation of 22 out of 25 nucleotides in an isotopically labelled 25-mer RNA. The results obtained with this new method are in agreement with the conformational analysis derived from ³J(H,H) coupling constants.     

Direct correlation of consecutive C′–N groups in proteins: a method for the assignment of intrinsically disordered proteins

Two novel 3D ¹³C-detected experiments, hNcocaNCO and hnCOcaNCO, are proposed to facilitate the resonance assignment of intrinsically disordered proteins. The experiments correlate the ¹⁵N and ¹³C′ chemical shifts of two consecutive amide moieties without involving other nuclei, thus taking advantage of the good dispersion shown by the ¹⁵N–¹³C′ correlations, even for proteins that lack a well defined tertiary structure. The new pulse sequences were successfully tested using Nupr1, an intrinsically disordered protein of 93 residues.     

Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy

Summary ³J _x coupling constants and complementary nuclear Overhauser data on the intraresidue C ^x H–CH distances form an essential part of the data needed to obtain stereospecific assignments of -methylene protons in proteins. In this paper we show that information regarding the magnitude of the³J _x coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D¹⁵N-separated¹H–¹H Hartmann-Hahn¹H–¹⁵N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue C ^x H–CH distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D¹³C-separated¹H–¹H rotating frame Overhauser effect¹H–¹³C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1, a protein of 153 residues and 17.4 kDa molecular weight.Abbreviations 1L-1 interleukin-1 - NOE nuclear Overhauser effect - ROE rotating frame Overhauser effect - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ROESY rotating frame Overhauser spectroscopy - HMQC heteronuclear multiple quantum coherence spectroscopy     

Oxidative stress–induced assembly of PML nuclear bodies controls sumoylation of partner proteins

The promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are stress-responsive domains where many partner proteins accumulate. Here, we clarify the basis for NB formation and identify stress-induced partner sumoylation as the primary NB function. NB nucleation does not rely primarily on intermolecular interactions between the PML SUMO-interacting motif (SIM) and SUMO, but instead results from oxidation-mediated PML multimerization. Oxidized PML spherical meshes recruit UBC9, which enhances PML sumoylation, allow partner recruitment through SIM interactions, and ultimately enhance partner sumoylation. Intermolecular SUMO–SIM interactions then enforce partner sequestration within the NB inner core. Accordingly, oxidative stress enhances NB formation and global sumoylation in vivo. Some NB-associated sumoylated partners also become polyubiquitinated by RNF4, precipitating their proteasomal degradation. As several partners are protein-modifying enzymes, NBs could act as sensors that facilitate and confer oxidative stress sensitivity not only to sumoylation but also to other post-translational modifications, thereby explaining alterations of stress response upon PML or NB loss.