NMR Chemical Shift Assignments Research Articles

Fluorine Patterning in Room-Temperature Fluorinated Graphite Determined by Solid-State NMR and DFT

Fluorination of graphite at room temperature allows producing graphite fluoride compounds with a controlled content of fluorine. Here we combine solid-state NMR spectroscopy and DFT calculations to study the structure and reveal the fluorine patterning in graphite fluorides C2Fx intercalated with acetonitrile. Two major chemical states of carbon, namely the atoms covalently bound to fluorine and the bare atoms, are detected by 13C MAS NMR irrespective of the degree of fluorination. The data indicate that although all graphene sheets were subjected to fluorination, the near-planar configuration is preserved. The interaction between host C2Fx matrix and acetonitrile molecules is of van der Waals character. Decomposition of the 19F MAS NMR spectra reveals occurrence of at least six fluorine environments in each sample. By DFT calculations distinct 19F chemical shifts are attributed to isolated, end chain, “linked” (which include midchain, cyclic, and branched) CF groups and infinite CF arrays. The assignment is confirmed by 19F RFDR, which is sensitive to dipolar coupling. Analyzing the data for C2Fx samples with different degrees of fluorination x, an evolution of the fluorine pattern is proposed. The reported calculated 19F NMR shielding parameters provide classification criteria for assignment of 19F NMR chemical shifts in fluorinated carbon materials.

Bacterial expression and purification of the amyloidogenic peptide PAPf39 for multidimensional NMR spectroscopy

PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enable structural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression and purification of uniformly 13C- and 15N-labeled PAPf39 peptide, through expression as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus. This allows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH.

1H and 13C NMR spectral assignments of an amino acid-coumarin hybrid

Synthesis and detailed spectral analysis of a new 4-substituted coumarin-amino acid derivate are presented in this paper. A new glycine derivate of (3-nitro-2-oxo-2H-chromen-4-yl)amino]acetate was prepared by condensation of 4-chloro-3-nitrocoumarin and ethyl glycinate hydrochloride. The complete assignment of 1H and 13C NMR chemical shifts of the synthesized compound was carried out by the aid of a combination of 1D (1H and 13C NMR) and 2D (1H-1H-COSY, NOESY, HSQC and HMBC) NMR experiments.

Plant cell wall profiling by fast maximum likelihood reconstruction (FMLR) and region-of-interest (ROI) segmentation of solution-state 2D 1H–13C NMR spectra

BackgroundInterest in the detailed lignin and polysaccharide composition of plant cell walls has surged within the past decade partly as a result of biotechnology research aimed at converting biomass to biofuels. High-resolution, solution-state 2D 1H–13C HSQC NMR spectroscopy has proven to be an effective tool for rapid and reproducible fingerprinting of the numerous polysaccharides and lignin components in unfractionated plant cell wall materials, and is therefore a powerful tool for cell wall profiling based on our ability to simultaneously identify and comparatively quantify numerous components within spectra generated in a relatively short time. However, assigning peaks in new spectra, integrating them to provide relative component distributions, and producing color-assigned spectra, are all current bottlenecks to the routine use of such NMR profiling methods.ResultsWe have assembled a high-throughput software platform for plant cell wall profiling that uses spectral deconvolution by Fast Maximum Likelihood Reconstruction (FMLR) to construct a mathematical model of the signals present in a set of related NMR spectra. Combined with a simple region of interest (ROI) table that maps spectral regions to NMR chemical shift assignments of chemical entities, the reconstructions can provide rapid and reproducible fingerprinting of numerous polysaccharide and lignin components in unfractionated cell wall material, including derivation of lignin monomer unit (S:G:H) ratios or the so-called SGH profile. Evidence is presented that ROI-based amplitudes derived from FMLR provide a robust feature set for subsequent multivariate analysis. The utility of this approach is demonstrated on a large transgenic study of Arabidopsis requiring concerted analysis of 91 ROIs (including both assigned and unassigned regions) in the lignin and polysaccharide regions of almost 100 related 2D 1H–13C HSQC spectra.ConclusionsWe show that when a suitable number of replicates are obtained per sample group, the correlated patterns of enriched and depleted cell wall components can be reliably and objectively detected even prior to multivariate analysis. The analysis methodology has been implemented in a publicly-available, cross-platform (Windows/Mac/Linux), web-enabled software application that enables researchers to view and publish detailed annotated spectra in addition to summary reports in simple spreadsheet data formats. The analysis methodology is not limited to studies of plant cell walls but is amenable to any NMR study where ROI segmentation techniques generate meaningful results.Please see Research Article: http://www.biotechnologyforbiofuels.com/content/6/1/46/.

¹H, ¹³C, and ¹⁵N chemical shift assignments of neuronal calcium sensor protein, hippocalcin.

Hippocalcin, a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, serves as an important calcium sensor for the slow afterhyperpolarizing (sAHP) current in the hippocampus, which underlies some forms of learning and memory. Hippocalcin is also a calcium sensor for hippocampal long-term depression (LTD) and genetically linked to neurodegenerative diseases. We report NMR chemical shift assignments of Ca(2+)-free hippocalcin (BMRB no. 18627).

1H, 13C and 15N resonance assignments of human BASP1

Brain acid-soluble protein 1 (BASP1, CAP-23, NAP-22) appears to be implicated in diverse cellular processes. An N-terminally myristoylated form of BASP1 has been discovered to participate in the regulation of actin cytoskeleton dynamics in neurons, whereas non-myristoylated nuclear BASP1 acts as co-suppressor of the potent transcription regulator WT1 (Wilms’ Tumor suppressor protein 1). Here we report NMR chemical shift assignment of recombinant human BASP1 fused to an N-terminal cleavable His6-tag.

1H, 13C and 15N resonance assignments of the complement control protein modules of the complement component C7

Human C7 is one of four homologous complement proteins that self-assemble on the nascent activation-specific fragment, C5b, thus forming the cytolytic membrane attack complex (MAC). In addition to the conserved modular core of the MAC/perforin protein family, C7 has four C-terminal domains comprising a pair of complement control protein modules (CCPs) preceding two Factor-I like modules (FIMs). It is proposed that the C7-CCPs might serve as a molecular arm for delivery of C7-FIMs to their binding site on C5b. Here we present the NMR chemical shift assignments for the C7-CCPs produced as a 14-kDa recombinant protein. Based upon triple-resonance experiments, 98 and 94 % of the backbone and side-chain ((1)H, (13)C and (15)N) assignments, respectively, have been completed. The chemical shifts and assignments have been deposited in the BioMagResBank database under accession number 18530.

Backbone (1)H, (13)C and (15)N NMR chemical shift assignments for the human DGCR8 core

Backbone (1)H, (13)C and (15)N NMR chemical shift assignments for the human DGCR8 core

Theoretical and Experimental Investigations on Hypoelectronic Heterodimetallaboranes of Group 6 Transition Metals

Density functional theory (DFT) has been used to probe the bonding and electronic properties of dimolybdaborane [(Cp*Mo)(2)B(5)H(9)], 1 (Cp* = η(5)-C(5)Me(5)), and several other heterodimolybdaborane clusters, such as [(Cp*Mo)(2)B(5)(μ(3)-OEt)H(7)] (2), [(Cp*Mo)(2)B(5)(μ(3)-OEt)(n-BuO)H(6)] (3), [(η(5)-C(5)H(5)W)(2)B(4)H(4)S(2)] (4), and [(Cp*Mo)(2)B(4)H(4)E(2)] (5-7, where, for 5, E = S, for 6, E = Se, and for 7, E = Te). The DFT results were also used to address some key points such as (i) the metal-metal bond length, (ii) the location and number of bridging and terminal hydrogen atoms, (iii) the molecular orbital analysis, and (iv) the assignment of (11)B and (1)H NMR chemical shifts. These studies further provide meticulous insight into similarities and differences between various dimetallaborane clusters 1-7. In addition, the crystal structures of 5 and 7 are reported, which come on top of the already existing literature of dimetallaboranes and support the theoretical findings.

A New Algorithm for Reliable and General NMR Resonance Assignment

The new FLYA automated resonance assignment algorithm determines NMR chemical shift assignments on the basis of peak lists from any combination of multidimensional through-bond or through-space NMR experiments for proteins. Backbone and side-chain assignments can be determined. All experimental data are used simultaneously, thereby exploiting optimally the redundancy present in the input peak lists and circumventing potential pitfalls of assignment strategies in which results obtained in a given step remain fixed input data for subsequent steps. Instead of prescribing a specific assignment strategy, the FLYA resonance assignment algorithm requires only experimental peak lists and the primary structure of the protein, from which the peaks expected in a given spectrum can be generated by applying a set of rules, defined in a straightforward way by specifying through-bond or through-space magnetization transfer pathways. The algorithm determines the resonance assignment by finding an optimal mapping between the set of expected peaks that are assigned by definition but have unknown positions and the set of measured peaks in the input peak lists that are initially unassigned but have a known position in the spectrum. Using peak lists obtained by purely automated peak picking from the experimental spectra of three proteins, FLYA assigned correctly 96-99% of the backbone and 90-91% of all resonances that could be assigned manually. Systematic studies quantified the impact of various factors on the assignment accuracy, namely the extent of missing real peaks and the amount of additional artifact peaks in the input peak lists, as well as the accuracy of the peak positions. Comparing the resonance assignments from FLYA with those obtained from two other existing algorithms showed that using identical experimental input data these other algorithms yielded significantly (40-142%) more erroneous assignments than FLYA. The FLYA resonance assignment algorithm thus has the reliability and flexibility to replace most manual and semi-automatic assignment procedures for NMR studies of proteins.

Backbone 1H, 13C, and 15N resonance assignments of guanylyl cyclase activating protein-1, GCAP1

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-dependent activation of retinal guanylyl cyclase that regulates the visual light response. GCAP1 is genetically linked to retinal degenerative diseases. We report backbone NMR chemical shift assignments of Ca(2+)-saturated GCAP1 (BMRB no. 18026).

Complete NMR assignment of 3, 4-seco -lup-20(29)-en-3-oic acid from Decatropis bicolor

Complete (1) H and (13) C NMR chemical shift assignments for 3,4-seco-lup-20(29)-en-3-oic acid (1) have been established by means of two-dimensional COSY, HSQC, HMBC and NOESY spectroscopic experiments as well as by analysis of MS data. Compound 1 was isolated from Decatropis bicolor (Zucc.) Radlk. (Rutaceae) in addition to six coumarins and one alkaloid of known structure.

1H, 15N, and 13C chemical shift assignments of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. We report complete NMR chemical shift assignments of the flagellar calcium binding protein calflagin Tb24 of Trypanosoma brucei. (BMRB no. 18011).

1H, 13C and 15N backbone and side chain resonance assignments of human halo S100A1

As part of our NMR structure determination of the Human S100A1, we report nearly complete NMR chemical shift assignments for the (1)H, (13)C and (15)N nuclei.

Other compounds isolated from Simira glaziovii and the ¹H and 13C NMR chemical shift assignments of new 1-epi-castanopsol

A new triterpene, 1-epi-castanopsol, besides eleven known compounds: sitosterol, stigmasterol, campesterol, lupeol, lupenone, simirane B, syringaresinol, scopoletin, isofraxidin, 6,7,8-trimethoxycoumarin and harman, were isolated from the wood of Simira glaziovii. The structures of the known compounds were defined by 1D, 2D ¹H, 13C NMR spectra data analyses and comparison with literature data. The detailed spectral data analyses allowed the definition of the structure of the new 1-epi isomer of castanopsol and performance of ¹H and 13C NMR chemical shift assignments.

Homo‐ and methano[60]fullerenes with chiral attached moieties – 1H and 13C NMR chemical shift assignments and diastereotopicity effects

(1)H and (13)C NMR chemical shift predictions of homo- and methano[60]fullerenes containing chiral centers in attached fragment were made using the two-dimensional NMR technique (HH COSY, (1)H-(13)C HSQC and HMBC) and the quantum chemistry GIAO calculation method in the PBE/3ζ approach. The influence of a chiral substituent on the (13)C chemical shifts of diastereotopic fullerene carbons was estimated by comparing the calculated and experimental (13)C NMR spectra. The resonances of the fullerene carbons in α-, β- and δ-positions relative to the position of the substituent exhibit the greatest diastereotopic splitting.

Fingerprints for Structural Defects in Poly(thienylene vinylene) (PTV): A Joint Theoretical–Experimental NMR Study on Model Molecules

In the field of plastic electronics, low band gap conjugated polymers like poly(thienylene vinylene) (PTV) and its derivatives are a promising class of materials that can be obtained with high molecular weight via the so-called dithiocarbamate precursor route. We have performed a joint experimental-theoretical study of the full NMR chemical shift assignment in a series of thiophene-based model compounds, which aims at (i) benchmarking the quantum-chemical calculations against experiments, (ii) identifying the signature of possible structural defects that can appear during the polymerization of PTV's, namely head-to-head and tail-to-tail defects, and (iii) defining a criterion regarding regioregularity.

Backbone and Ile-δ1, Leu, Val Methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein

Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone (1)H(N), (15)N, (13)C', and (13)C(α), as well as side chain (13)C(β), methyl (Ile-δ1, Leu, Val), amide (Asn, Gln), and indole N-H (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.

Complete 1H and 13C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses 1H and 13C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the 1H and 13C, as well as 31P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 1H and 13C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D 1H, 13C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t 1 incremented 1H, 13C-HSQC experiment and a 1D 1H, 1H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3 Hz apart. The 1H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program ( http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental 1H and 13C NMR chemical shifts.

NMR assignments of ubiquitin fold domain (UFD) in SUMO-activating enzyme subunit 2 from rice

The small ubiquitin-related modifier (SUMO) is a ubiquitin-like post-translational modifier that alters the localization, activity, or stability of many proteins. In the sumoylation process, an activated SUMO is transferred from SUMO-activating enzyme E1 complex (SAE1/SAE2) to SUMO-conjugating enzyme E2 (Ubc9). Among the multiple domains in E1, a C-terminal ubiquitin fold domain (UFD) of SAE2 shows high affinity for Ubc9, implying that UFD will be functionally important. We report NMR chemical shift assignments of UFD in SAE2 from rice. Almost all the resonances of UFD were assigned uniquely, representing a single conformation of UFD in solution. This is a contrast to the previous report for the corresponding UFD of human SAE2 which shows two conformational states. The secondary structure prediction of UFD in rice SAE2 shows the similar overall structure to the crystal structures of UFD in other E1 proteins such as SAE2 of human and yeast, ubiquitin-activating enzyme of yeast, and NEDD8-activating enzyme E1 catalytic subunit of human. Concomitantly, differences in the length of helices, strands, and loops are observed, particularly in the binding region to E2, supposing the variation in the UFD-E2 binding mode which may play a critical role in determining E1-E2 specificity.