Solanopubamine,a steroidal alkaloid from Solanum pubescens

Aerial parts of Solanum pubescens yielded a new steroidal alkaloid, solanopubamine, the structure of which was elucidated as 3β-amino-5α,22αH,25βH-solanidan-23β-ol by ¹³C NMR, ¹H NMR, IR, mass spectral analysis and chemical degradation methods.     

Withanolide A series steroidal lactones from Eucalyptus globulus bark

A new steroidal lactone of the Withanolide A series has been isolated from the supercritical fluid extract of Eucalyptus globulus L. (bark) as a major component (I) along with a known structurally similar steroidal lactone as minor component (II). The structural identification of the new lactone was accomplished by different spectroscopic techniques viz. ¹H and ¹³C NMR, etc. The relative stereochemistry was unequivocally determined from the X-ray crystallography.     

(25R)-Isonuatigenin,an unusual steroidal sapogenin from Vestia lycioides

(25R)-Isonuatigenin, a ⁵-spirosten-3β,25-diol, was isolated from aerial parts of Vestia lycioides. Its structure was elucidated mainly by ¹H NMR and ¹³C NMR spectroscopy. A mixture of (25R)-nuatigenin and (25S)-isonuatigenin was also characterized. This is the first report on the natural occurrence of the two (25R)-isomers.     

Quantitative analysis of metabolite concentrations in human urine samples using 13C{1H} NMR spectroscopy

Targeted profiling is a library-based method of using mathematically modeled reference spectra for quantification of metabolite concentrations in NMR mixture analysis. Metabolomics studies of biofluids, such as urine, represent a highly complex problem in this area, and for this reason targeted profiling of ¹H NMR spectra can be hampered. A number of the issues relating to ¹H NMR spectroscopy can be overcome using ¹³C{¹H} NMR spectroscopy. In this work, a ¹³C{¹H} NMR database was created using Chenomx NMR Suite, incorporating 120 metabolites. The ¹³C{¹H} NMR database was standardized through the analysis of a series of metabolite solutions containing varying concentrations of 19 distinct metabolites, where the metabolite concentrations were varied across a range of values including biological ranges. Subsequently, the NMR spectra of urine samples were collected using ¹³C{¹H} NMR spectroscopy and profiled using the ¹³C{¹H} NMR library. In total, about 30 metabolites were conclusively identified and quantified in the urine samples using ¹³C{¹H} NMR targeted profiling. The proton decoupling and larger spectral window provided easier identification and more accurate quantification for specific classes of metabolites, such as sugars and amino acids with overlap in the aliphatic region of the ¹H NMR spectrum. We discuss potential application areas in which ¹³C{¹H} NMR targeted profiling may be superior to ¹H NMR targeted profiling.     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state C NMR studies on [3-C]Ala- and [1-C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full ¹³C NMR signals from whole area of proteins. Well-resolved ¹³C NMR signals were recorded for monomeric [3-¹³C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several ¹³C NMR signals from the loops and transmembrane α-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10⁴-10⁵ Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the ¹³C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 °C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for ¹³C NMR observation. It is therefore concluded that [3-¹³C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-¹³C]Ala- and [1-¹³C]Val-bR at low temperature gave rise to well-resolved ¹³C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.     

Innovations in Generation and Analysis of 2D [C,H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

2D [¹³C,¹H] COSY NMR is used by the metabolic engineering community for determining ¹³C–¹³C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [¹³C,¹H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range ¹³C–¹³C scalar couplings in 2D [¹³C,¹H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Magic-angle-spinning solid-state ¹³C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-¹³C,¹⁵N labeled samples. To overcome this problem, we present a method for assigning ¹³C chemical shifts and secondary structures from unresolved two-dimensional ¹³C–¹³C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts and cross-peak intensities. The experimental ¹³C–¹³C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C^α, C^β, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved ¹³C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.     

C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence-total correlation spectroscopy (HMQC-TOCSY)

¹³C has become an important tracer isotope for studies of intermediary metabolism. Information about relative flux through pathways is encoded by the distribution of ¹³C isotopomers in an intermediate pool such as glutamate. This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a ¹³C NMR spectrum. We demonstrate here that groups of glutamate ¹³C isotopomers may be quantified by indirect detection of protons in a 2D HMQC-TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the ¹³C fractional enrichment of acetyl-CoA and relative anaplerotic flux to that given by direct ¹³C NMR analysis. The sensitivity gain provided by HMQC-TOCSY spectroscopy will allow an extension of ¹³C isotopomer analysis to tissue samples not amenable to direct ¹³C detection (∼10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C,15N-labeled RNAs

¹³C spin diluted protein samples can be produced using [1-¹³C] and [2-¹³C]-glucose (Glc) carbon sources in the bacterial growth medium. The ¹³C spin dilution results in favorable ¹³C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-¹³C]- and [2-¹³C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-¹³C]-glycerol (¹³C labeled Cα sites on a ¹²C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17–26, 2011 ). Inspired by this approach and our own recent results using [2-¹³C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of ¹³C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.     

Base-type-selective high-resolution 13C edited NOESY for sequential assignment of large RNAs

Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully ¹³C labelled RNA. High-resolution ¹H-¹³C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear ¹³C decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any ¹³C-edited multidimensional NMR experiment. As an example we have chosen the ¹³C-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.     

An NMR method for the determination of protein binding interfaces using TEMPOL-induced chemical shift perturbations

Background

The determination of protein–protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein–protein interface by NMR spectroscopy, ¹³C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct ¹³C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while ¹³C nuclei within the interior of the proteins may be distinguished by a lack of such shifts.

Method

We measured the ¹³C NMR paramagnetic shifts induced by TEMPOL by recording ¹³C–¹³C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1).

Results

Upon complexation of ubiquitin with YUH1, ¹³C NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data.

Conclusions

The TEMPOL-induced ¹³C chemical shift perturbation is useful to determine precise protein–protein interfaces.

General significance

The present method is a useful method to determine protein–protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities.     

Synthesis of steroid-ferrocene conjugates of steroidal 17-carboxamides via a palladium-catalyzed aminocarbonylation--copper-catalyzed azide-alkyne cycloaddition reaction sequence

Steroids with the 17-iodo-16-ene functionality were converted to ferrocene labeled steroidal 17-carboxamides via a two step reaction sequence. The first step involved the palladium-catalyzed aminocarbonylation of the alkenyl iodides with prop-2-yn-1-amine as the nucleophile in the presence of the Pd(OAc)₂/PPh₃ catalyst system. In the second step, the product N-(prop-2-ynyl)-carboxamides underwent a facile azide–alkyne cycloaddition with ferrocenyl azides in the presence of CuSO₄/sodium ascorbate to produce the steroid–ferrocene conjugates. The new compounds were obtained in good yield and were characterized by ¹H and ¹³C NMR, IR, MS and elemental analysis.     

NMR assignment of the nonstructural protein nsp3(1066–1181) from SARS-CoV

Sequence-specific NMR assignments of the globular core comprising the residues 1066–1181 within the non-structural protein nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [¹³C, ¹⁵N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure determination. A preliminary identification of regular secondary structures has been derived from the ¹³C chemical shifts.     

High resolution nmr and x-ray crystallography data of caudicifolin from Euphorbia acaulis

A diterpene lactone was isolated from the cold petrol (60−80°) extract of rhizomes of Euphorbia acaulis, a plant material used by a tribe of central India for curing various inflammatory disorder. The diterpene, which was observed to be identical to caudicifolin on the basis of its physical constants, was subjected to high resolution NMR spectroscopy and X-ray crystallography examination. This paper reports the salient features of the 2D ¹H NMR, ¹³C NMR and X-ray crystallography data of the compound. ¹³C NMR assignments were made by the use of proton noise decoupling, SFORD, APT and automatic spectral editing techniques. ¹H NMR assignments were made with the aid of a COSY experiment for long range couplings and NOE correlated 2D-experiments. The ¹H and ¹³C NMR spectral assignments have been further corroborated by H/C correlation experimental results.     

Natural Abundance Fourier Transform of <Superscript>13</Superscript>C Nuclear Magnetic Resonance Spectra of Lysozyme

NUCLEAR magnetic resonance (NMR) studies of biopolymers have so far been limited to proton resonances. On the other hand, ¹³C NMR analysis is potentially a more powerful technique because of the larger range of chemical shifts (300 p.p.m. compared with 10 p.p.m. for ^XH NMR) and because the chemical shift of ¹³C NMR is more sensitive to the nature of the chemical bonds. The chief limitations of ¹³C NMR are low natural abundance (1·1%), low relative sensitivity (1·59% of proton) and long relaxation times (?1 min). These can be largely overcome, however, by the combined techniques of nuclear Overhauser enhancement and fourier transform spectroscopy. Thus, Gibbons et al.¹ obtained a simple and elegant spectral analysis of ‘Gramicidin S-A’, a decapeptide. We report here the first natural abundance ¹³C NMR spectrum of an enzyme. Lysozyme was chosen because its primary structure is known^2,3 and because its ¹H NMR spectrum has already been described⁴.     

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.     

A fast NMR method for resonance assignments: application to metabolomics

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (¹²C) or ¹³C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [¹³C, ¹H] HSQC–TOCSY, (2) 2D ¹H–¹H TOCSY and (3) 2D ¹³C–¹H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [¹³C, ¹H] HSQC–TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete ¹H and ¹³C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.     

Fungal macrolides: Structure determination and biosynthesis of achaetolide,a lactone from Achaetomium cristalliferum

The structure of a new ten-membered lactone, achaetolide, isolated from cultures of Achaetomium cristalliferum is deduced from its mass and NMR spectra and from the study ofsomederivatives. The ¹³C NMR spectra of achaetolide enriched with [1-¹³C], [2-¹³C] and [1, 2-¹³C] acetate established its formation from eight intact acetate units via a precursor octaketide chain.     

An acyclic diterpene from the brown alga Bifurcaria bifurcata

Structure elucidation and total assignment of the ¹³C NMR spectrum of 12-(S)-hydroxygeranylgeraniol, a new acyclic diterpene from the grown alga Bifurcaria bifurcata, was accomplished through the use of ¹HNMR, ¹³C NMR and 2D NMR spectroscope including 2D long range ¹H-¹³C chemical shift correlations.