Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins

Natively unfolded proteins are increasingly recognized to play important physiological roles. These proteins do not crystallize, so NMR is the only technique able to provide structural and dynamic information. However, in unfolded proteins, the proton chemical shift dispersion is poor, causing severe problems in resonance assignment. We designed a novel strategy based on two protonless experiments, a CBCACON-IPAP and a novel COCON-IPAP, that permits a straightforward and unequivocal backbone heteronuclear assignment of the natively unfolded protein alpha-synuclein.     

Biomolecular NMR using a microcoil NMR probe--new technique for the chemical shift assignment of aromatic side chains in proteins

A specially designed microcoil probe for use in biomolecular NMR spectroscopy is presented. The microcoil probe shows a mass-based sensitivity increase of a minimal factor of 7.5, allowing for the first time routine biomolecular NMR spectroscopy with microgram amounts of proteins. In addition, the exceptional radio frequency capabilities of this probe allowed us to record an aliphatic-aromatic HCCH-TOCSY spectrum for the first time. Using this spectrum, the side chains of aliphatic and aromatic amino acids can be completely assigned using only a single experiment. Using the conserved hypothetical protein TM0979 from Thermotoga maritima, we demonstrate the capabilities of this microcoil NMR probe to completely pursue the sequence specific backbone assignment with less than 500 microg of (13)C,(15)N labeled protein.     

Heteronuclear selective refocusing 2D NMR experiments for the spectral analysis of enantiomers in chiral oriented solvents

We report the use of carbon-proton heteronuclear selective refocusing 2D NMR experiments dedicated to the spectral analysis of enantiomers dissolved in weakly ordering chiral liquid crystalline solvents. The method permits the extraction of carbon-proton residual dipolar couplings for each enantiomer from a complex or unresolved proton-coupled 13C spectral patterns. Illustrative examples are analysed and discussed. It is shown that an accurate determination of enantiomeric excess is possible.     

Automatic assignment of 13C NMR spectra based on the chemical shift/charge density relationship

An approach for the automatic assignment of ¹³C spectra, based on the chemical shift/charge density relationship, is suggested. All permutations of spectral signals are computer-generated, and for every permutation a least squares adjustment is carried out. The permutation presenting the highest correlation coefficient, or the lowest Hamilton's agreement factor, is considered to be correct. The application is exemplified by the ¹³C chemical shifts of a series of aromatic compounds. It is shown that more reliable assignment is achieved if the considered permutations are restricted by taking intoaccount the signal multiplicity.     

二维NMR技术在有机结构分析中的应用

联合应用二维远程碳 -氢相关 (coloc)技术和二维碳 -氢相关(hxdept)技术 ,对三个含季碳较多的化合物进行了谱峰归属与结构指定。从而显示出多维NMR技术在有机结构分析中的重要作用。     

Structural and spectral assignment by 2D NMR of a new prenylated benzopyrancarboxylic acid and structural reassignment of a related compound

A new prenylated benzopyrancarboxylic acid, 1a (3,4‐dihydro‐5‐hydroxy‐2,7‐dimethyl‐8‐(2‐methyl‐2‐butenyl)‐ 2‐(4‐methyl‐1, 3‐pentadienyl)‐2H‐1‐benzopyran‐6‐carboxylic acid) was isolated from Peperomia amplexicaulis and fully characterized by 1D and 2D NMR and high‐resolution mass spectrometry. In the course of this investigation, the structure of a related compound (minus the carboxylic acid group) which was previously assigned as 2b was corrected to structure 1b . Copyright © 2003 John Wiley & Sons, Ltd.     

Semiautomatic sequence-specific assignment of proteins based on the tertiary structure--the program st2nmr

The sequence-specific assignment of resonances is still the most time-consuming procedure that is necessary as the first step in high-resolution NMR studies of proteins. In many cases a reliable three-dimensional (3D) structure of the protein is available, for example, from X-ray spectroscopy or homology modeling. Here we introduce the st2nmr program that uses the 3D structure and Nuclear Overhauser Effect spectroscopy (NOESY) peak list(s) to evaluate and optimize trial sequence-specific assignments of spin systems derived from correlation spectra to residues of the protein. A distance-dependent target function that scores trial assignments based on the presence of expected NOESY crosspeaks is optimized in a Monte Carlo fashion. The performance of the program st2nmr is tested on real NMR data of an alpha-helical (cytochrome c) and beta-sheet (lipocalin) protein using homology models and/or X-ray structures; it succeeded in completely reproducing the correct sequence-specific assignments in most cases using 2D and/or 15N/13C Nuclear Overhauser Effect (NOE) data. Additionally to amino acid residues the program can also handle ligands that are bound to the protein, such as heme, and can be used as a complementary tool to fully automated assignment procedures.     

一维多重接力COSY和选择性远程DEPT核磁共振技术在新分子骨架生物碱结构测定和谱峰归属研究中的应用

自防已科千金藤根中分得一个新型分子骨架的四氢异喹啉类生物碱-excentricine(1)。本工作采用一维多重接力COSY和选择性远程DEPT核磁共振新技术成功地确定谱峰归属, 鉴别和连结被季碳和杂原子分割开的自旋体系, 测定了其结构。     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments VI: chemical shift analysis of his to gain 3D structure and protonation state information

NMR--chemical shift structure correlations were investigated by using GIAO-RB3LYP/6-311++G(2d,2p) formalism. Geometries and chemical shifts (CSI values) of 103 different conformers of N'-formyl-L-histidinamide were determined including both neutral and charged protonation forms. Correlations between amino acid torsional angle values and chemical shifts were investigated for the first time for an aromatic and polar amino acid residue whose side chain may carry different charges. Linear correlation coefficients of a significant level were determined between chemical shifts and dihedral angles for CSI[(1)H(alpha)]/phi, CSI[(13)C(alpha)]/phi, and CSI[(13)C(alpha)]/psi. Protonation of the imidazole ring induces the upfield shift of CSI[(13)C(alpha)] for positively charged histidines and an opposite effect for the negative residue. We investigated the correspondence of theoretical and experimental (13)C(alpha), (13)C(beta), and (1)H(alpha) chemical shifts and the nine basic conformational building units characteristic for proteins. These three chemical shift values allow the identification of conformational building units at 80% accuracy. These results enable the prediction of additional regular secondary structural elements (e.g., polyProlineII, inverse gamma-turns) and loops beyond the assignment of chemical shifts to alpha-helices and beta-pleated sheets. Moreover, the location of the His residue can be further specified in a beta-sheet. It is possible to determine whether the appropriate residue is located at the middle or in a first/last beta-strand within a beta-sheet based on calculated CSI values. Thus, the attractive idea of establishing local residue specific backbone folding parameters in peptides and proteins by employing chemical shift information (e.g., (1)H(alpha) and (13)C(alpha)) obtained from selected heteronuclear correlation NMR experiments (e.g., 2D-HSQC) is reinforced.     

Assignment of 1H signals in complex overlapping spectral regions using proton-detected 2D heteronuclear shift correlation spectroscopy

The application of a heteronuclear 2D pulse sequence is demonstrated which correlates chemical shifts of carbons with directly bound protons and with remote protons belonging to the same coupled spin system by using the effect of Hartmann—Hahn cross polarization among protons. The enhanced spectral resolution obtained by spreading individual proton subsystems into the carbon domain and the option for multiplicity discrimination makes these kind of experiments most suitable for proton and carbon signal assignments of natural products as alkaloids and steroids with strongly overlapping proton resonances. With the application to a steroid we want to demonstrate that such unambiguous signal assignments are the prerequisite and the basis for detailed structural investigations using additional, more conventional NMR experiments.     

Identifying residues in natural organic matter through spectral prediction and pattern matching of 2D NMR datasets

This paper describes procedures for the generation of 2D NMR databases containing spectra predicted from chemical structures. These databases allow flexible searching via chemical structure, substructure or similarity of structure as well as spectral features. In this paper we use the biopolymer lignin as an example. Lignin is an important and relatively recalcitrant structural biopolymer present in the majority of plant biomass. We demonstrate how an accurate 2D NMR database of approximately 600 2D spectra of lignin fragments can be easily constructed, in approximately 2 days, and then subsequently show how some of these fragments can be identified in soil extracts through the use of various search tools and pattern recognition techniques. We demonstrate that once identified in one sample, similar residues are easily determined in other soil extracts. In theory, such an approach can be used for the analysis of any organic mixtures.     

Structure assignment in the solid state by the coupling of quantum chemical calculations with NMR experiments: a columnar hexabenzocoronene derivative.

We present a quantum chemical ab initio study which demonstrates a new combined experimental and theoretical approach, whereby a comparison of calculated and experimental (1)H NMR chemical shifts allows the elucidation of structural arrangements in solid-state molecular ensembles, taking advantage of the marked sensitivity of the (1)H chemical shift to intermolecular interactions. Recently, Brown et al. have shown that, under fast magic-angle spinning (MAS) at 35 kHz, the resolution in a (1)H NMR spectrum of the solid phase of an alkyl-substituted hexabenzocoronene (HBC) derivative is sufficient to observe the hitherto unexpected resolution of three distinct aromatic resonances ( J. Am. Chem. Soc. 1999, 121, 6712). Exploiting the additional information about proton proximities provided by (1)H double-quantum (DQ) MAS NMR spectroscopy, it was shown that the results are qualitatively consistent with the aromatic cores packing in a manner similar to that in unsubstituted HBC. Using the HBC-C(12) molecule as an example, we show here that the new combined experimental and theoretical approach allows the observed (1)H chemical shifts to be related in a quantitative manner to the intermolecular structure. In the quantum chemical calculations, a series of model systems of stacked HBC oligomers are used. On account of the marked dependence of the (1)H chemical shift to ring currents arising from nearby aromatic rings, the calculated (1)H chemical shifts are found to be very sensitive to the stacking arrangement of the HBC molecules. Moreover, the ring current effect is found to be particularly long range, with a considerable influence of the second neighbor, at a distance of 700 pm, being observed.     

NMR spectra of pyrimidines effect of substituents on the chemical shift of the para protons in 2- and 5-substituted pyrimidines

The chemical shifts of the protons of the pyrimidine ring in the 2 position for a series of 5-substituted pyrimidines and in the 5 position for a series of 2-substituted pyrimidines in solutions in dimethyl sulfoxide were determined. The correlation equations that link the relative chemical shifts with the F and R substituent constants were calculated. The correlation equations were analyzed by comparison with the corresponding correlation equation for a series of monosubstituted benzenes. The reasons for the change in the conductivity of the electronic effects of the substituents via inductive and conjugation mechanisms in the pyrimidine ring as compared with the conductivity in the benzene ring are discussed.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 4, pp. 526–529, April, 1978.     

Stereochemistry and conformations of natural 1,2-epoxy-guaianolides based on 1D and 2D NMR data and semiempirical calculations

From detailed study of 1D and 2D NMR spectra of ten natural 1,2-epoxyguaianolides (bis-1,2:3,4-epoxyguaianolides and guaianolide-1,2-epoxychlorohydrins), we identified general spectral traits helpful for stereochemical assignment of such sesquiterpene lactones. We found that the chemical shifts of certain (1)H and (13)C nuclei are consistently dependent on the configuration of 1,2-epoxy-ring which could be used as a simple rule for establishing this configuration. Then, from 1D and 2D (COSY, NOESY, HMQC, HMBC) NMR data, applying the observed rule, the structure and stereochemistry of two new, diastereomeric guaianolide-1,2-epoxychlorohydrins, isolated from Achillea serbica, are determined. The NMR data, namely, nuclear overhauser enhancement (NOE) correlations, pointed out two conformations of guaianolide's cycloheptane ring. The semiempirical calculations (AM1 and PM3 methods), performed in order to gain additional information regarding conformations, resulted in three geometries of investigated lactones. Even so, the conformations derived from the NMR data agreed well with those calculated by semiempirical methods.     

Stereospecific assignments of protein NMR resonances based on the tertiary structure and 2D/3D NOE data

In many cases of protein structure determination by NMR a high-quality structure is required. An important contribution to structural precision is stereospecific assignment of magnetically nonequivalent prochiral methylene and methyl groups, eliminating the need for introducing pseudoatoms and pseudoatom corrections in distance restraint lists. Here, we introduce the stereospecific assignment program that uses the resonance assignment, a preliminary 3D structure and 2D and/or 3D nuclear Overhauser effect spectroscopy peak lists for stereospecific assignment. For each prochiral group the algorithm automatically calculates a score for the two different stereospecific assignment possibilities, taking into account the presence and intensity of the nuclear Overhauser effect (NOE) peaks that are expected from the local environment of each prochiral group (i.e., the close neighbors). The performance of the algorithm has been tested and used on NMR data of alpha-helical and beta-sheet proteins using homology models and/or X-ray structures. The program produced no erroneous stereospecific assignments provided the NOEs were carefully picked and the 3D model was sufficiently accurate. The set of NOE distance restraints produced by nmr2st using the results of the SSA module was superior in generating good-quality ensembles of NMR structures (low deviations from upper limits in conjunction with low root-mean-square-deviation values) in the first round of structure calculations. The program uses a novel approach that employs the entire 3D structure of the protein to obtain stereospecific assignment; it can be used to speed up the NMR structure refinement and to increase the quality of the final NMR ensemble even when no scalar or residual dipolar coupling information is available.     

Near-Threshold and Resonance Effects in Rotationally Inelastic Scattering of D2O with Normal-H2

We present a combined experimental and theoretical study on the rotationally inelastic scattering of heavy water, D₂O, with normal-H₂. Crossed-molecular beam measurements are performed in the collision energy range between 10 and 100 cm⁻¹, corresponding to the near-threshold regime in which scattering resonances are most pronounced. State-to-state excitation cross-sections are obtained by probing three low-lying rotational levels of D₂O using the REMPI technique. These measurements are complemented by quantum close-coupling scattering calculations based on a high-accuracy D₂O–H₂ interaction potential. The agreement between experiment and theory is within the experimental error bars at 95% confidence intervals, leading to a relative difference of less than 7%: the near-threshold rise and the overall shape of the cross-sections, including small undulations due to resonances, are nicely reproduced by the calculations. Isotopic effects (D₂O versus H₂O) are also discussed by comparing the shape and magnitude of the respective cross-sections.     

Discrimination and analysis of the NMR spectra of enantiomers dissolved in chiral liquid crystal solvents through 2D correlation experiments

The discrimination and analysis of the NMR spectra of optically active molecules dissolved in chiral liquid crystal solvents through 2D correlation experiments is studied. The technique allows the identification of the line positions of each enantiomer, thus providing a notable simplification of the spectral analysis. The 2D HOHAHA and multiple-quantum experiments are investigated and discussed. The potential of the method is illustrated using a sample of (±) 3,3,3-trichloroepoxypropane dissolved in a thermotropic cholesteric solvent. The case of chiral molecules bearing a fluorine or deuterium nucleus has also been studied. In addition, it is shown that 2D heteronuclear correlation experiments are powerful methods for correlating carbon and proton spectral data of two enantiomers. A specific example is given through (±) 2-bromopropanoic acid dissolved in a lyotropic polypeptide liquid crystal. Spectral parameters of each enantiomer are calculated for the different examples.     

胃泌素有关肽溶液构象的2D-NMR研究

用2D NMR深入研究了五肽、四肽胃泌素在 DMSO 中的构象。利用 COSY 谱、Relayed-COSY 谱、DQF-J 分解谱和 NOESY 谱归属了全部共振峰。计算了肽键平面的Φ角、旋转异构体分布和旋转异构体之间自由能差。根据 NOESY 谱得到了距离约束条件。结合Φ角及旋转异构体分布, 推导出五肽胃泌素分子的构象膜型。结果表明, 五肽、四肽胃泌素在 DMSO 中以半角构象存在, 在其主要的旋转异构体中 Trp 的吲哚环和 Met 的 S 原子具有类似5,1-benzothiazocine 三维结构, 可能是具有生物活性的原因。     

Spectrometric and 2D NMR Studies on the Complexation of Chlorophenols with Cyclodextrins

The formation and structure of inclusion complexes of - and-cyclodextrins with 2-chlorophenol (2CP), 3-chlorophenol (3CP),4-chlorophenol (4CP), 2,4-dichlorophenol (24DCP), 2,6-dichlorophenol(26DCP) and 3,4-dichlorophenol (34DCP) have been studied by UV-VIS and¹H NMR spectroscopy. Both cyclodextrins were found to form 1:1inclusion complexes. Bindingconstants estimated from titration studies revealed that the stability of the complexes was highly dependent on the structure and polarity of the chlorophenol and on the cyclodextrin used. In general, weaker binding constants were observed for a given chlorophenol with -cyclodextrin than with-cyclodextrin. The weakest binding constants (K_{b < 200} M^-1) were obtained for the ortho-substituted chlorophenols (2CP and 26DCP) and the largest binding constants were obtained between para-chlorophenols (4CP, 24DCP and 34DCP) and-cyclodextrin. 2D-TROESY studies of chlorophenol-cyclodextrincomplexes in D₂O provided insight into the structure of the complexes.     

The anisotropic effect of 4-substituents on the 1H NMR chemical shift of H-5 in phenanthrenes

Chloro-, methoxy- and methyl-substituents in the 4-position of phenanthrenes exert a profound and diagnostic influence on the ¹H chemicalshift of H-5, and from ¹³C measurements it is considered to be dueto an anisotropic effect.