SANE (Structure Assisted NOE Evaluation): An automated model-based approach for NOE assignment

A reliable automated approach for assignment of NOESY spectra would allow more rapid determination of protein structures by NMR. In this paper we describe a semi-automated procedure for complete NOESY assignment (SANE, Structure Assisted NOE Evaluation), coupled to an iterative procedure for NMR structure determination where the user is directly involved. Our method is similar to ARIA [Nilges et al. (1997) J. Mol. Biol., 269, 408–422], but is compatible with the molecular dynamics suites AMBER and DYANA. The method is ideal for systems where an initial model or crystal structure is available, but has also been used successfully for ab initio structure determination. Use of this semi-automated iterative approach assists in the identification of errors in the NOE assignments to short-cut the path to an NMR solution structure.     

Interproton distance bounds from 2D NOE intensities: Effect of experimental noise and peak integration errors

Summary The effect of experimental and integration errors on the calculations in interproton distances from NOE intensities is examined. It is shown that NOE intensity errors can have a large impact on the distances determined. When multiple spin (spin diffusion) effects are significant, the calculated distances are often underestimated, even when using a complete relaxation matrix analysis. In this case, the bias of distances to smaller values is due to the random errors in the NOE intensities. We show here that accurate upper and lower bounds of the distances can be obtained if the intensity errors are properly accounted for in the complete relaxation matrix calculations, specifically the MARDIGRAS algorithm. The basic MARDIGRAS algorithm has been previously described [Borgias, B.A. and James, T.L. (1990) J. Magn. Reson., 87, 475–487]. It has been shown to provide reasonably good interproton distance bounds, but experimental errors can compromise the quality of the resulting restraints, especially for weak cross peaks. In a new approach introduced here, termed RANDMARDI (random error MARDIGRAS), errors due to random noise and integration errors are mimicked by the addition of random numbers from within a specified range to each input intensity. Interproton distances are then calculated for the modified intensity set using MARDIGRAS. The distribution of distances that define the upper and lower distance bounds is obtained by using N randomly modified intensity sets. RANDMARDI has been used in the solution structure determination of the interstrand cross-link (XL) formed between 4-hydroxymethyl-4,5,8-trimethylpsoralen (HMT) and the DNA oligomer d(5-GCGTACGC-3)₂ [Spielmann, H.P. et al. (1995) Biochemistry, 34, 12937–12953]. RANDMARDI generates accurate distance bounds from the experimental NOESY cross-peak intensities for the fixed (known) interproton distances in XL. This provides an independent internal check for the ability of RANDMARDI to accurately fit the experimental data. The XL structure determined using RANDMARDI-generated restrains is in good agreement with other biophysical data that indicate that there is no bend introduced into the DNA by the cross-link. In contrast, isolated spin-pair approximation calculations give distance restraints that, when applied in a restrained molecular dynamics protocol, produce a bent structure.Abbreviations NOE nuclear Overhauser effect - SD standard deviation - HMT 4-hydroxymethyl-4,5,8-trimethylpsoralen - XL psoralen-DNA interstrand cross-link     

Complete relaxation matrix refinement of NMR structures of proteins using analytically calculated dihedral angle derivatives of NOE intensities

Summary A new method for refining three-dimensional (3D) NMR structures of proteins is described, which takes account of the complete relaxation pathways. Derivatives of the NOE intensities with respect to the dihedral angles are analytically calculated, and efficiently evaluated with the use of a filter technique for identifying the dominant terms of these derivatives. This new method was implemented in the distance geometry program DIANA. As an initial test, we refined 30 rigid distorted helical structures, using a simulated data set of NOE distance constraints for a rigid standard -helix. The final root-mean-square deviations of the refined structures relative to the standard helix were less than 0.1 Å, and the R-factors dropped from values between 7% and 32% to values of less than 0.5% in all cases, which compares favorably with the results from distance geometry calculations. In particular, because spin diffusion was not explicitly considered in the evaluation of exact¹H–¹H distances corresponding to the simulated NOE intensities, a group of nearly identical distance geometry structures was obtained which had about 0.5 Å root-mean-square deviation from the standard -helix. Further test calculations using an experimental NOE data set recorded for the protein trypsin inhibitor K showed that the complete relaxation matrix refinement procedure in the DIANA program is functional also with systems of practical interest.Abbreviations RMSD root-mean-square deviation - NOE nuclear Overhauser enhancement - NOESY 2-dimensional nuclear Overhauser enhancement spectroscopy - CPU central processing unit     

Extensive distance geometry calculations with different NOE calibrations: New criteria for structure selection applied to Sandostatin and BPTI

Summary To generate structures efficiently, a version of the distance geometry program DIANA for a parallel computer was developed, new objective criteria for the selection of NMR solution structures are presented, and the influence of using different calibrations of NOE intensities on the final structures are described. The methods are applied to the structure determination of Sandostatin, a disulfide-bridge octapeptide, and to model calculations of BPTI. On an Alliant FX2800 computer using 10 processors in parallel, the calculations were done 9.2 times faster than with a single processor. Up to 7000 Sandostatin structures were calculated with distance and angular constraints. The procedure for selecting acceptable structures is based on the maximum values of pairwise RMSDs between structures. Suitable target function cut-offs are defined independent of the number of starting structures. The method allowed for an objective comparison of three groups of Sandostatin structures that were calculated from different sets of upper distance constraints which were derived from the same NOE intensity data using three empirical calibration curves. The number of converged structures and the target function values differed significantly among the three groups, but the structures were qualitatively and quantitatively very similar. The conformation is well determined in the cyclic region Cys²–Cys⁷ and adopts a -turn centered at d-Trp⁴–Lys⁵. The criteria for structure selection were further tested with BPTI. Results obtained from sets of structures calculated with and without using the REDAC strategy are consistent and suggest that the structure selection method is objective and generally applicable.     

The influence of internuclear spatial distribution and instrument noise on the precision of distances determined by solid state NMR of isotopically enriched proteins

The relative merits of different isotopic enrichment strategies that might be used in solid state NMR protein structure determinations are explored. The basis for comparison of these merits is the determination of the relative uncertainties in rates measured by a generalized dipolar recoupling experiment. The different schemes considered use ¹³C, ¹⁵N and ²H labeling of ubiquitin with homonuclear magnetization-transfer type experiments under magic-angle spinning (MAS). Specific attention is given to the sensitivity of the predicted relative precisions to variation in natural nuclear density distribution and noise levels. A framework is suggested to gauge the precision of measurement of a given dipolar coupling constant, and the potential for a set of such measurements to constrain structure calculations is explored. The distribution of nuclei in homonuclear ¹⁵N and ¹H dipolar recoupling spin-exchange experiments appear to provide the most promising tertiary structure information for uniformly labeled ubiquitin.     

Structural and dynamic studies of two antigenic loops from haemagglutinin: A relaxation matrix approach

Summary We have investigated the dynamics and structural behaviour of two antigenic peptides using ¹H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a -turn in both structures.Abbreviations DSS 3-(trimethylsilyl)-1-propanesulfonic acid - D-loop aspartic acid loop - ELISA enzyme-linked immunoabsorbent assay - f.i.d free induction decay - HOHAHA homonuclear Hartmann-Hahn spectroscopy - HPLC high pressure liquid chromatography - K-loop lysine loop - NMR nuclear magnetic resonance - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - r.m.s.d. root-mean-square deviation of atomic positions     

Influence of the completeness of chemical shift assignments on NMR structures obtained with automated NOE assignment

Reliable automated NOE assignment and structure calculation on the basis of a largely complete, assigned input chemical shift list and a list of unassigned NOESY cross peaks has recently become feasible for routine NMR protein structure calculation and has been shown to yield results that are equivalent to those of the conventional, manual approach. However, these algorithms rely on the availability of a virtually complete list of the chemical shifts. This paper investigates the influence of incomplete chemical shift assignments on the reliability of NMR structures obtained with automated NOESY cross peak assignment. The program CYANA was used for combined automated NOESY assignment with the CANDID algorithm and structure calculations with torsion angle dynamics at various degrees of completeness of the chemical shift assignment which was simulated by random omission of entries in the experimental ¹H chemical shift lists that had been used for the earlier, conventional structure determinations of two proteins. Sets of structure calculations were performed choosing the omitted chemical shifts randomly among all assigned hydrogen atoms, or among aromatic hydrogen atoms. For comparison, automated NOESY assignment and structure calculations were performed with the complete experimental chemical shift but under random omission of NOESY cross peaks. When heteronuclear-resolved three-dimensional NOESY spectra are available the current CANDID algorithm yields in the absence of up to about 10% of the experimental ¹H chemical shifts reliable NOE assignments and three-dimensional structures that deviate by less than 2 Å from the reference structure obtained using all experimental chemical shift assignments. In contrast, the algorithm can accommodate the omission of up to 50% of the cross peaks in heteronuclear- resolved NOESY spectra without producing structures with a RMSD of more than 2 Å to the reference structure. When only homonuclear NOESY spectra are available, the algorithm is slightly more susceptible to missing data and can tolerate the absence of up to about 7% of the experimental ¹H chemical shifts or of up to 30% of the NOESY peaks.Abbreviations: BmPBP^A – Bombyx mori pheromone binding protein form A; CYANA – combined assignment and dynamics algorithm for NMR applications; NMR – nuclear magnetic resonance; NOE – nuclear Overhauser effect; NOESY – NOE spectroscopy; RMSD – root-mean-square deviation; WmKT – Williopsis mrakii killer toxin     

Automated NMR determination of protein backbone dihedral angles from cross-correlated spin relaxation

The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei ¹³C, ¹H,¹³CO, ¹⁵N and ¹H^N can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state ¹³CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between ¹H(i)-¹³C(i)/¹⁵N(i)-¹H^N(i) and ¹H(i–1)-¹³C(i–1)/¹⁵N(i)-¹H^N(i) dipole-dipole mechanisms in ¹⁵N,¹³C-labeled proteins. The approach is illustrated with an application to ¹³C,¹⁵N-labeled ubiquitin.     

Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cgamma-Hgamma/Cbeta-Hbeta residual dipolar couplings: application to the 34-kDa enzyme IIA(chitobiose)

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a ¹⁵N/¹³C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)–H_N(i) and Hγ(i)–H_N(i+1) NOEs in a 3D ¹⁵N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine ¹³C–¹H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA^Chitobiose, a 34 kDa homotrimeric phosphotransferase protein.     

PGSE diffusion NMR studies on mononuclear and dinuclear cationic platinum salts of (S)-MeO-Biphep and (R)-p-tolyl-BINAP

PGSE diffusion NMR studies on a series of mononuclear and dinuclear cationic platinum salts derived from (S)-MeO-Biphep and (R)-p-tolyl-BINAP are reported. The data show that (a) one can readily distinguish between mononuclear and dinuclear cations (b) the amount of ion pairing can be estimated qualitatively and (c) the charge delocalization rather than the amount of formal charge per metal cation is important for the position of the anion. The solid-state structure of the chloro-bridged salt, [Pt(μ-Cl){(S)-MeO-Biphep}]₂(CF₃SO₃)₂, is reported.     

H, F-HOESY and PGSE diffusion studies on ionic liquids: The effect of co-solvent on structure

¹H, ¹⁹F-HOESY studies on the ionic liquid based on 1-butyl-3-methylimidazolium tetrafluoroborate, [C₄C₁im]BF₄, [C₄C₁im][N(OTf)₂], and, partially, [C₄C₁im]PF₆, are reported. The addition of methanol separates the ions; however, in dichloromethane solution the anions and cations show strong HOESY contacts even after dilution and taken together with the PGSE diffusion measurements, the data suggest strong ion pairing in this solvent.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without ¹⁵N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties.     

Accounting for uncertainty in the tree topology has little effect on the decision-theoretic approach to model selection in phylogeny estimation

Currently available methods for model selection used in phylogenetic analysis are based on an initial fixed-tree topology. Once a model is picked based on this topology, a rigorous search of the tree space is run under that model to find the maximum-likelihood estimate of the tree (topology and branch lengths) and the maximum-likelihood estimates of the model parameters. In this paper, we propose two extensions to the decision-theoretic (DT) approach that relax the fixed-topology restriction. We also relax the fixed-topology restriction for the Bayesian information criterion (BIC) and the Akaike information criterion (AIC) methods. We compare the performance of the different methods (the relaxed, restricted, and the likelihood-ratio test [LRT]) using simulated data. This comparison is done by evaluating the relative complexity of the models resulting from each method and by comparing the performance of the chosen models in estimating the true tree. We also compare the methods relative to one another by measuring the closeness of the estimated trees corresponding to the different chosen models under these methods. We show that varying the topology does not have a major impact on model choice. We also show that the outcome of the two proposed extensions is identical and is comparable to that of the BIC, Extended-BIC, and DT. Hence, using the simpler methods in choosing a model for analyzing the data is more computationally feasible, with results comparable to the more computationally intensive methods. Another outcome of this study is that earlier conclusions about the DT approach are reinforced. That is, LRT, Extended-AIC, and AIC result in more complicated models that do not contribute to the performance of the phylogenetic inference, yet cause a significant increase in the time required for data analysis.     

The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Gα_i proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Gα_i protein activity appears to significantly reduce Hh pathway activity in Ptc^−/− mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Gα_i protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Gα_i protein, Gα_i2QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Gα_i activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.     

Bmi-1 over-expression in neural stem/progenitor cells increases proliferation and neurogenesis in culture but has little effect on these functions in vivo

The polycomb gene Bmi-1 is required for the self-renewal of stem cells from diverse tissues, including the central nervous system (CNS). Bmi-1 expression is elevated in most human gliomas, irrespective of grade, raising the question of whether Bmi-1 over-expression is sufficient to promote self-renewal or tumorigenesis by CNS stem/progenitor cells. To test this we generated Nestin-Bmi-1-GFP transgenic mice. Analysis of two independent lines with expression in the fetal and adult CNS demonstrated that transgenic neural stem cells formed larger colonies, more self-renewing divisions, and more neurons in culture. However, in vivo, Bmi-1 over-expression had little effect on CNS stem cell frequency, subventricular zone proliferation, olfactory bulb neurogenesis, or neurogenesis/gliogenesis during development. Bmi-1 transgenic mice were born with enlarged lateral ventricles and a minority developed idiopathic hydrocephalus as adults, but none of the transgenic mice formed detectable CNS tumors, even when aged. The more pronounced effects of Bmi-1 over-expression in culture were largely attributable to the attenuated induction of p16^Ink4a and p19^Arf in culture, proteins that are generally not expressed by neural stem/progenitor cells in young mice in vivo. Bmi-1 over-expression therefore has more pronounced effects in culture and does not appear to be sufficient to induce tumorigenesis in vivo.