Backbone-only restraints for fast determination of the protein fold: the role of paramagnetism-based restraints. Cytochrome b562 as an example

CH(alpha) residual dipolar couplings (Deltardc's) were measured for the oxidized cytochrome b562 from Escherichia coli as a result of its partial self-orientation in high magnetic fields due to the anisotropy of the overall magnetic susceptibility tensor. Both the low spin iron (III) heme and the four-helix bundle fold contribute to the magnetic anisotropy tensor. CH(alpha) Deltardc's, which span a larger range than the analogous NH values (already available in the literature) sample large space variations at variance with NH Deltardc's, which are largely isooriented within alpha helices. The whole structure is now significantly refined with the chemical shift index and CH(alpha) Deltardc's. The latter are particularly useful also in defining the molecular magnetic anisotropy parameters. It is shown here that the backbone folding can be conveniently and accurately determined using backbone restraints only, which include NOEs, hydrogen bonds, residual dipolar couplings, pseudocontact shifts, and chemical shift index. All these restraints are easily and quickly determined from the backbone assignment. The calculated backbone structure is comparable to that obtained by using also side chain restraint. Furthermore, the structure obtained with backbone only restraints is, in its whole, very similar to that obtained with the complete set of restraints. The paramagnetism based restraints are shown to be absolutely relevant, especially for Deltardc's.     

Correction of spin diffusion during iterative automated NOE assignment

Indirect magnetization transfer increases the observed nuclear Overhauser enhancement (NOE) between two protons in many cases, leading to an underestimation of target distances. Wider distance bounds are necessary to account for this error. However, this leads to a loss of information and may reduce the quality of the structures generated from the inter-proton distances. Although several methods for spin diffusion correction have been published, they are often not employed to derive distance restraints. This prompted us to write a user-friendly and CPU-efficient method to correct for spin diffusion that is fully integrated in our program ambiguous restraints for iterative assignment (ARIA). ARIA thus allows automated iterative NOE assignment and structure calculation with spin diffusion corrected distances. The method relies on numerical integration of the coupled differential equations which govern relaxation by matrix squaring and sparse matrix techniques. We derive a correction factor for the distance restraints from calculated NOE volumes and inter-proton distances. To evaluate the impact of our spin diffusion correction, we tested the new calibration process extensively with data from the Pleckstrin homology (PH) domain of Mus musculus beta-spectrin. By comparing structures refined with and without spin diffusion correction, we show that spin diffusion corrected distance restraints give rise to structures of higher quality (notably fewer NOE violations and a more regular Ramachandran map). Furthermore, spin diffusion correction permits the use of tighter error bounds which improves the distinction between signal and noise in an automated NOE assignment scheme.     

Editing of Chemical Exchange-Relayed NOEs in NMR Experiments for the Observation of Protein–Water Interactions

An experimental approach for the editing of exchange-relayed NOEs in water-selective NOE experiments is presented. The proposed pulse sequence is based on the application during the NOE mixing time of continuous wave irradiation, which saturates resonances of relaying labile protons in slow chemical exchange with water. The technique can efficiently reduce the contributions of exchange-relayed NOE peaks that often crowd the water-selective NOE spectra and hide direct intermolecular NOEs between water and protein protons. The present approach opens new opportunities for the characterization of hydration by NMR, even in the proximity of polar labile groups.     

Determining a helical protein structure using peptide pixels

The residual dipolar coupling-periodicity planarity correlation makes it possible to determine peptide plane orientations in regular periodic protein secondary structure elements. Each peptide plane orientation represents a "pixel" of protein structure, and is expressed in terms of three angles referred to as tilt, phase, and pitch angles. In this report, we present the novel "3P" (periodicity, planarity, and pixels) method that allows one to determine secondary and tertiary structure of alpha-helical proteins. We demonstrate the 3P method by determining the structure of domain 1 of the receptor-associated protein (RAP) to a backbone accuracy of 1.0 Angstrom using RDCs measured in a single alignment medium, together with a minimal number of NOE distance restraints, using a new Xplor-NIH module.     

Selective Correlation of Amide Groups to Glycine Alpha Protons and of Arginine Guanidine Groups to Delta Protons in Proteins by Multiple Quantum Spectroscopy

A new 3D pulse sequence correlates backbone amide proton and nitrogen with alpha proton resonances selectively for glycine residues in a fully doubly labeled (¹⁵N,¹³C) protein. The excitation of multiple quantum coherences provides optimized resolution and sensitivity. Degenerate alpha proton groups can be promptly recognized. Correlation of guanidine NH groups to delta protons of arginine side chains is also obtained.     

Asymmetric MRI magnet design using a hybrid numerical method

Two-dimensional 1H/13C polarization inversion spin exchange at the magic angle experiments were applied to single crystal samples of amino acids to demonstrate their potential utility on oriented samples of peptides and proteins. High resolution is achieved and structural information obtained on backbone and side chain sites from these spectra. A triple-resonance experiment that correlates the 1H-13Calpha dipolar coupling frequency with the chemical shift frequencies of the alpha-carbon, as well as the directly bonded amide 15N site, is also demonstrated. In this experiment the large 1H-13Calpha heteronuclear dipolar interaction provides an independent frequency dimension that significantly improves the resolution among overlapping 13C resonances of oriented polypeptides, while simultaneously providing measurements of the 13Calpha chemical shift, 1H-13C dipolar coupling, and 15N chemical shift frequencies and angular restraints for backbone structure determination.     

Two-Dimensional Transferred Nuclear-Overhauser Effects with Incomplete Averaging of Free- and Bound-Ligand Resonances

Two distinct time scales prevail in the study of conformational exchange in general and specifically in deriving the protein-bound conformations of ligands by two-dimensional transferred-NOE experiments. Fast-exchange compared to the chemical-shift difference leads to a complete averaging of the free and bound resonances of the ligands, while fast exchange compared to the longitudinal relaxation rate 1/T₁ and cross relaxation results in an average of the relaxation matrices in the free and bound states. Slower chemical exchange leads to incomplete averaging of the free and bound resonances, giving rise to a reduction of transferred NOEs compared to fast-exchange conditions, Theoretical analysis and simulation results indicated that, in the absence of information on the chemical-shift time scale, transferred NOEs may be quantitated only to an accuracy with average NOE intensities and transferred NOEs to the free ligands as upper and lower bounds, respectively. Regardless of the exchange conditions, NOE intensities can be approximated by the NOEs transferred to the free ligands when a large excess of the free ligands (α_b < 0.1) is used to amplify the NOE transfers between the bound protons, These results suggest that two-dimensional transferred-NOE experiments (transferred NOESY) can be applied in situations of intermediate and slow exchange as long as the fraction of bound ligands is small and the exchange rates are fast enough to transfer the cross-relaxation pathways in the protein-ligand complex to the easily detected resonances of the free ligands,     

NMR assignment of protein side chains using residue-correlated labeling and NOE spectra

A new approach for the isotopic labeling of proteins is proposed that aims to facilitate side chain resonance assignments. Residue-correlated (RC) labeling is achieved by the expression of a protein on a medium containing a mixture of labeled, e.g., [U-13C,15N]amino acids, and NMR silent, [U-2H]amino acids. De novo synthesis of amino acids was suppressed by feedback inhibition by the amino acids in the growth medium and by the addition of beta-chloro-L-alanine, a transaminase inhibitor. Incorporation of these amino acids into synthesized proteins results in a relative diminution of inter-residue NOE interactions and a relative enhancement of intra-residue NOEs. Comparison of the resulting NOE spectra with those obtained from a uniformly labeled sample allows identification of intra-residue NOE peaks. Thus, this approach provides direct information for sidechain assignments in the NOE spectra, which are subsequently used for structural analysis. We have demonstrated the feasibility of this strategy for the 143 amino acid nuclease inhibitor NuiA, both at 35 degrees C, corresponding to a rotational correlation time of 9.5 ns, and at 5 degrees C, corresponding to a rotational correlation time of 22 ns.     

Automatic assignment of NOESY cross peaks and determination of the protein structure of a new world scorpion neurotoxin using NOAH/DIAMOD

The 3D NMR structures of the scorpion neurotoxin, CsE-v5, were determined from the same NOESY spectra with NOAH/DIAMOD, an automated assignment and 3D structure calculation software package, and with a conventional manual assignment combined with a distance geometry/simulated annealing (X-PLOR) refinement method. The NOESY assignments and the 3D structures obtained from the two independent methods were compared in detail. The NOAH/DIAMOD program suite uses feedback filtering and self-correcting distance geometry methods to automatically assign NOESY spectra and to calculate the 3D structure of a protein. NOESY cross peaks were automatically picked using a standard software package and combined with 74 manually assigned NOESY peaks to start the NOAH/DIAMOD calculations. After 63 NOAH/DIAMOD cycles, using REDAC procedures in the last 8 cycles, and final FANTOM constrained energy minimization, a bundle of 20 structures with the smallest target functions has a RMSD of 0.81 A for backbone atoms and 1.11 A for all heavy atoms to the mean structure. Despite some missing chemical shifts of side chain protons, 776 (including 74 manually assigned) of 1130 NOE peaks were unambiguously assigned, 150 peaks have more than one possible assignment compatible with the bundle structures, and only 30 peaks could not be assigned within the given chemical shift tolerance ranges in either the D1 or the D2 dimension. The remaining 174, mainly weak NOE peaks were not compatible with the final 20 best bundle structures at the last NOAH/DIAMOD cycle. The automatically determined structures agree well with the structures determined independently using the conventional method and the same NMR spectra, with the mean RMSD in well-defined regions of 0.84 A for bb and 1.48 A for all heavy atoms from residues 2-5, 18-26, 32-36, and 39-45. This study demonstrates the potential of the NOAH/DIAMOD program suite to automatically assign NMR data for proteins and determine their structure.     

Refined solution structure of bovine pancreatic Ribonuclease A by1H NMR methods. Sidechain dynamics

An extension of the assignment of the¹H NMR spectra of bovine pancreatic Ribonuclease A has been carried out at 600 MHz covering all sidechain protons, including those belonging to long sidechain residues. Vicinal coupling constants,³ J _HNα and³ J _αβ and³ J _αβ′ have been measured with the purpose of imposing torsional constraints on the backbone ? angle as well as to introduceH _ββ′ specific assignments. On the basis of distance constraints evaluated from 1285 assigned NOEs, a first restrained molecular dynamics calculation has been carried out. The resulting root-mean-square deviation for the backbone atoms is 1.5 Å as compared to 1.8 Å for the 360 MHz NOE basis. For heavy atoms in sidechains, these numbers were, respectively, 3.0 and 2.5 Å A second calculation was carried out by introducing 37 dihedral angle constraints on backbone ?, torsions and five onX ₁ angular torsions as well as with stereospecific assignments for nearly 50 residues. Backbone and sidechain RMS deviations were, respectively, 1.2 and 2.0 Å. Sidechains have been classified in terms of their dynamical behavior aroundX ₁ after considering jointly the coupling information and that resulting from the second set of calculated structures. Results are discussed in relation to the crystal structure.     

Protein dynamics measurements by TROSY-based NMR experiments

The described TROSY-based experiments for investigating backbone dynamics of proteins make it possible to elucidate internal motions in large proteins via measurements of T(1), T(2), and NOE of backbone (15)N nuclei. In our proposed sequences, the INEPT sequence is eliminated and the PEP sequence is replaced by the ST2-PT sequence from the HSQC-based experiments. This has the benefit of shortening the pulse sequences by 5.4 ms (=1/2J) and results in an increase in the intrinsic sensitivity of the proposed TROSY-based experiments. The TROSY-based experiments are on average of 13% more sensitive than the corresponding HSQC-based experiments on a uniformly (15)N-labeled Xenopus laevis calcium-bound calmodulin sample on a 750-MHz spectrometer at 5 degrees C. The amide proton linewidths of the TROSY-based experiments are 2-13 Hz narrower than those of the HSQC experiments. More sensitivity gain and higher resolution are expected if the protein sample is deuterated.     

多肽和蛋白质NMR预饱和实验中饱和转移效应的研究

通过改变预饱和照射时间研究了一些多肽和蛋白质分子在水溶液中的饱和转移效应.定量分析了在照射60s之后大分子中绝大多数酰胺质子仍不受影响的原因.结果表明:具有稳定的三维溶液结构的蛋白质(如溶菌酶)中,活泼氢的信号基本上不受饱和转移影响;对于溶液结构比较稳定的多肽和蛋白质(如胰岛素),只有少部分酰胺质子信号强度受到影响;小分子六肽因为溶液中不存在稳定的构象,饱和转移效应十分显著.因此对于溶液中多肽与蛋白质构象的NMR研究,预饱和仍不失为一种简便而有效的溶剂峰压制技术.     

Intermolecular (1)H[(19)F] NOEs in studies of fluoroalcohol-induced conformations of peptides and proteins

Mixtures of fluorinated alcohols and water can selectively stabilize certain secondary structures of peptides and proteins. Such mixtures may also be of use in solubilizing hydrophobic or membrane-bound proteins. We show that intermolecular dipolar interactions between the fluorine nuclei of such solvents and the protons of a dissolved protein lead to readily detected (1)H[(19)F] nuclear Overhauser effects. These NOEs can potentially provide information about solvent exposure of particular groups as well as indicate the formation of long-lived fluoroalcohol-solute complexes. Results obtained with HEW lysozyme in solutions containing trifluoroethanol illustrate these possibilities.     

人体胰岛素质子交换反应的核磁共振研究

用线型拟合的方法通过核磁共振谱图测得了人体胰岛素R6六聚体中酰胺质子与重氢的交换速度.因为用这一方法对实验谱图进行计算模拟时考虑了核磁共振谱线所提供的所有信息,所以我们获得了化学位移非常接近的蛋白质酰胺质子与重氢的交换速度,这是用其它方法难以得到的数据.结果表明,用本文所述方法获得的酰胺质子的交换速度与由2DNMR计算所得的蛋白质结构有非常好的吻合.在获得酰胺质子交换速度的基础上,本文详细讨论了交换反应速度与蛋白质结构的关系,结果表明,在该蛋白质中影响酰胺质子交换速度大小的因素以强弱顺序排列为:质子在肽链中的位置 > 是否参与形成氢键 > 蛋白质的二级及三级结构.     

The fumarate sensor DcuS: progress in rapid protein fold elucidation by combining protein structure prediction methods with NMR spectroscopy

We illustrate how moderate resolution protein structures can be rapidly obtained by interlinking computational prediction methodologies with un- or partially assigned NMR data. To facilitate the application of our recently described method of ranking and subsequent refining alternative structural models using unassigned NMR data [Proc. Natl. Acad. Sci. USA 100 (2003) 15404] for such "structural genomics"-type experiments it is combined with protein models from several prediction techniques, enhanced to utilize partial assignments, and applied on a protein with an unknown structure and fold. From the original NMR spectra obtained for the 140 residue fumarate sensor DcuS, 1100 1H, 13C, and 15N chemical shift signals, 3000 1H-1H NOESY cross peak intensities, and 209 backbone residual dipolar couplings were extracted and used to rank models produced by de novo structure prediction and comparative modeling methods. The ranking proceeds in two steps: first, an optimal assignment of the NMR peaks to atoms is found for each model independently, and second, the models are ranked based on the consistency between the NMR data and the model assuming these optimal assignments. The low-resolution model selected using this ranking procedure had the correct overall fold and a global backbone RMSD of 6.0 angstrom, and was subsequently refined to 3.7 angstrom RMSD. With the incorporation of a small number of NOE and residual dipolar coupling constraints available very early in the traditional spectral assignment process, a model with an RMSD of 2.8 angstrom could rapidly be built. The ability to generate moderate resolution models within days of NMR data collection should facilitate large scale NMR structure determination efforts.     

NMR spectroscopy reveals common structural features of the birch pollen allergen Bet v 1 and the cherry allergen Pru a 1

A high percentage of birch pollen allergic patients also experience food hypersensitivity which results from common epitopes on the corresponding allergens. In order to analyze whether this observed cross-reactivity can be attributed to common structural features, the major birch pollen allergen Bet v 1 and the cherry allergen Pru a 1 were investigated by multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. For the 17 kDa Bet v 1 a three-dimensional structure was calculated on the basis of 1344 experimental restraints. The structure is well defined showing average root mean square deviations of 0.67 and 1.15 Å for the backbone heavy atoms and all heavy atoms of residues 1–154, respectively. The major structural features include a seven-stranded antiparallel β-sheet that wraps around a long C-terminal α-helix, thereby forming a large cavity in the interior of the protein. This structure served as template for the generation of an NMR-based model of Pru a 1 by homology modelling in conjunction with 277 experimentally derived distance restraints. Comparison to the structure of Bet v 1 proves both structures to be highly similar concerning the elements of secondary structure as well as the shape and charge distribution of the protein surface. This finding is consistent with the observed cross-reactivity between both proteins and allows the delineation of common cross-reactive B-cell epitopes.     

EPR和NMR自旋标记法研究部分折叠蛋白质构象

部分折叠蛋白质的特征是在溶液中有部分二级结构,但是三级结构接触比较松散或者较少,其意义可能是蛋白质折叠过程的中间态,或者有重要的生理功能. 利用EPR和NMR波谱可以表征其构象特征,从而构建其二级结构、三级结构、四级结构以及构象变化的结构模型. 利用分子生物学的点突变技术可以在蛋白质主链上插入1个或2个半胱氨酸残基,然后把顺磁自旋标记物特异地共价结合在半胱氨酸的侧链巯基上来制备自旋标记样品. 位点特异性自旋标记电子顺磁共振（SDSL-EPR）波谱是通过测定2个自旋标记间的偶极相互作用,从而推测2个硝基氧自由基间的距离. 核磁共振（NMR）波谱则是通过测定单个自旋标记中心对周围原子核驰豫效应增强（PRE）的效应,推测出顺磁中心相对于周围原子核的距离. 连续波EPR和NMR自旋标记方法可以测定2.5 nm左右的偶极相互作用距离,属于长程的距离约束,这对于确定部分折叠蛋白质的构象至关重要. 该文将就蛋白质部分折叠态研究的生物学意义,自旋标记方法以及EPR和NMR方法研究其构象特征举例描述.     

Hydrogen bonds in human ubiquitin reflected in temperature coefficients of amide protons

Analysis of amide proton temperature coefficients (deltasigma(HN)/DeltaT) in human ubiquitin shows their usefulness in indicating hydrogen bonds. The availability of a very accurate solution structure of ubiquitin enables the precise determination of hydrogen bonds and increases the reliability of the analysis of chemical shift temperature gradients. Values of deltasigma(HN)/DeltaT more positive than -4.6 ppb/K are very good indicators of hydrogen bonds. Additionally, a weak temperature dependence of non-hydrogen-bonded amides was observed for amide protons that are significantly shifted upfield. We observed that temperature gradients of amide protons involved in short hydrogen bonds are related to donor-acceptor distances.     

Vibrational spectra and crystal structure of the di‐amino acid peptide cyclo(L‐Met‐L‐Met): comparison of experimental data and DFT calculations

Experimental Raman and FT‐IR spectra of solid‐state non‐deuterated and N‐deuterated samples of cyclo(L ‐Met‐L ‐Met) are reported and discussed. The Raman and FT‐IR results show characteristic amide I vibrations (Raman: 1649 cm⁻¹, infrared: 1675 cm⁻¹) for molecules exhibiting a cis amide conformation. A Raman band, assigned to the cis amide II vibrational mode, is observed at ∼1493 cm⁻¹ but no IR band is observed in this region. Cyclo(L ‐Met‐L ‐Met) crystallises in the triclinic space group P1 with one molecule per unit cell. The overall shape of the diketopiperazine (DKP) ring displays a (slightly distorted) boat conformation. The crystal packing employs two strong hydrogen bonds, which traverse the entire crystal via translational repeats. B3‐LYP/cc‐pVDZ calculations of the structure of the molecule predict a boat conformation for the DKP ring, in agreement with the experimentally determined X‐ray structure. Copyright © 2009 John Wiley & Sons, Ltd.     

Phenomenological nuclear-reaction description in deuterium-saturated palladium and synthesized structure in dense deuterium gas under γ-quanta irradiation

The observed phenomena of changes of chemical compositions in previous reports [1, 2] allowed us to develop a phenomenological nuclear fusion-fission model with taking into consideration the elastic and inelastic scattering of photoprotons and photoneutrons, heating of surrounding deuterium nuclei, following d-d fusion reactions and fission of middle-mass nuclei by “hot” protons, deuterons and various-energy neutrons. Such chain processes could produce the necessary number of neutrons, “hot” deuterons for explanation of observed experimental results [1, 2]. The developed approach can be a basis for creation of deuterated nuclear fission reactors (DNFR) with high-density deuterium gas and the so-called deuterated metals. Also, this approach can be used for the study of nuclear reactions in high-density deuterium or tritium gas and deuterated metals.