Identification Of Spin Systems Research Articles

Demonstration of NV-detected ESR spectroscopy at 115 GHz and 4.2 T

High frequency electron spin resonance (ESR) spectroscopy is an invaluable tool for identification and characterization of spin systems. Nanoscale ESR using the nitrogen-vacancy (NV) center has been demonstrated down to the level of a single spin. However, NV-detected ESR has exclusively been studied at low magnetic fields, where the spectral overlap prevents clear identification of spectral features. In this work, we demonstrate NV-detected ESR measurements of single-substitutional nitrogen impurities in diamond at a NV Larmor frequency of 115 GHz and the corresponding magnetic field of 4.2 T. The NV-ESR measurements utilize a double electron-electron resonance sequence and are performed using both ensemble and single NV spin systems. In the single NV experiment, chirp pulses are used to improve the population transfer and for NV-ESR measurements. This work provides the basis for NV-based ESR measurements of external spins at high magnetic fields.

ASAP-HSQC-TOCSY for fast spin system identification and extraction of long-range couplings.

Based on Ernst-angle-type excitation and Acceleration by Sharing Adjacent Polarization (ASAP), a fast HSQC-TOCSY experiment is introduced. In the approach, the DIPSI-2 isotropic mixing period of the ASAP-HSQC is simply shifted, which provides a TOCSY period without additional application of rf-energy. The ASAP-HSQC-TOCSY allows the acquisition of a conventional 2D in about 30 s. Alternatively, it allows the acquisition of highly carbon-resolved spectra (several Hz digital resolution) on the order of minutes. An ASAP-HSQC-TOCSY-IPAP variant, finally, allows the sign-sensitive extraction of heteronuclear long-range coupling constants from a pair of highly resolved spectra in less than an hour. Pulse sequences, several example spectra, and a discussion of results are given.

Non-Uniform and Absolute Minimal Sampling for High-Throughput Multidimensional NMR Applications.

Many biomolecular NMR applications can benefit from the faster acquisition of multidimensional NMR data with high resolution and their automated analysis and interpretation. In recent years, a number of non-uniform sampling (NUS) approaches have been introduced for the reconstruction of multidimensional NMR spectra, such as compressed sensing, thereby bypassing traditional Fourier-transform processing. Such approaches are applicable to both biomacromolecules and small molecules and their complex mixtures and can be combined with homonuclear decoupling (pure shift) and covariance processing. For homonuclear 2D TOCSY experiments, absolute minimal sampling (AMS) permits the drastic shortening of measurement times necessary for high-throughput applications for identification and quantification of components in complex biological mixtures in the field of metabolomics. Such TOCSY spectra can be comprehensively represented by graphic theoretical maximal cliques for the identification of entire spin systems and their subsequent query against NMR databases. Integration of these methods in webservers permits the rapid and reliable identification of mixture components. Recent progress is reviewed in this Minireview.

Chemical shift-based identification of monosaccharide spin-systems with NMR spectroscopy to complement untargeted glycomics.

A better understanding of oligosaccharides and their wide-ranging functions in almost every aspect of biology and medicine promises to uncover hidden layers of biology and will support the development of better therapies. Elucidating the chemical structure of an unknown oligosaccharide remains a challenge. Efficient tools are required for non-targeted glycomics. Chemical shifts are a rich source of information about the topology and configuration of biomolecules, whose potential is however not fully explored for oligosaccharides. We hypothesize that the chemical shifts of each monosaccharide are unique for each saccharide type with a certain linkage pattern, so that correlated data measured by NMR spectroscopy can be used to identify the chemical nature of a carbohydrate. We present here an efficient search algorithm, GlycoNMRSearch, which matches either a subset or the entire set of chemical shifts of an unidentified monosaccharide spin system to all spin systems in an NMR database. The search output is much more precise than earlier search functions and highly similar matches suggest the chemical structure of the spin system within the oligosaccharide. Thus, searching for connected chemical shift correlations within all electronically available NMR data of oligosaccharides is a very efficient way of identifying the chemical structure of unknown oligosaccharides. With an improved database in the future, GlycoNMRSearch will be even more efficient deducing chemical structures of oligosaccharides and there is a high chance that it becomes an indispensable technique for glycomics. The search algorithm presented here, together with a graphical user interface, is available at http://glyconmrsearch.nmrhub.eu. Supplementary data are available at Bioinformatics online.

Application of Dirichlet process mixture model to the identification of spin systems in protein NMR spectra

Analysis of structure, function and interactions of proteins by NMR spectroscopy usually requires the assignment of resonances to the corresponding nuclei in protein. This task, although automated by methods such as FLYA or PINE, is still frequently performed manually. To facilitate the manual sequence-specific chemical shift assignment of complex proteins, we propose a method based on Dirichlet process mixture model (DPMM) that performs automated matching of groups of signals observed in NMR spectra to corresponding nuclei in protein sequence. The model has been extensively tested on 80 proteins retrieved from the BMRB database and has shown superior performance to the reference method.

Maximal clique method for the automated analysis of NMR TOCSY spectra of complex mixtures.

Characterization of the chemical components of complex mixtures in solution is important in many areas of biochemistry and chemical biology, including metabolomics. The use of 2D NMR total correlation spectroscopy (TOCSY) experiments has proven very useful for the identification of known metabolites as well as for the characterization of metabolites that are unknown by taking advantage of the good resolution and high sensitivity of this homonuclear experiment. Due to the complexity of the resulting spectra, automation is critical to facilitate and speed-up their analysis and enable high-throughput applications. To better meet these emerging needs, an automated spin-system identification algorithm of TOCSY spectra is introduced that represents the cross-peaks and their connectivities as a mathematical graph, for which all subgraphs are determined that are maximal cliques. Each maximal clique can be assigned to an individual spin system thereby providing a robust deconvolution of the original spectrum for the easy extraction of critical spin system information. The approach is demonstrated for a complex metabolite mixture consisting of 20 compounds and for E. coli cell lysate.

GFT projection NMR for efficient 1H/13C sugar spin system identification in nucleic acids

A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify (1)H/(13)C sugar spin systems in (13)C labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of (13)C-(1)H groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1'- and 3'-CH signal dispersion for complete spin system identification including 5'-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3'-untranslated region of the pre-mRNA of human U1A protein.

Speeding up sequence specific assignment of IDPs

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

Self-Consistent Metabolic Mixture Analysis by Heteronuclear NMR. Application to a Human Cancer Cell Line

Elucidation of the chemical composition of biological samples is a main focus of systems biology and metabolomics. In order to comprehensively study these complex mixtures, reliable, efficient, and automatable methods are needed to identify and quantify the underlying metabolites and natural products. Because of its rich information content, nuclear magnetic resonance (NMR) spectroscopy has a unique potential for this task. Here we present a generalization of the recently introduced homonuclear TOCSY-based DemixC method to heteronuclear HSQC-TOCSY NMR spectroscopy. The approach takes advantage of the high resolution afforded along the (13)C dimension due to the narrow (13)C line widths for the identification of spin systems and compounds. The method combines information from both 1D (13)C and (1)H traces by querying them against an NMR spectral database using our COLMAR query web server. The complementarity of (13)C and (1)H spectral information improves the robustness of compound identification. The method is demonstrated for a metabolic model mixture and is then applied to an extract from DU145 human prostate cancer cells.

Study of angiotensin-(1–7) vasoactive peptide and its β-cyclodextrin inclusion complexes: Complete sequence-specific NMR assignments and structural studies

We report the complete sequence-specific hydrogen NMR assignments of vasoactive peptide angiotensin-(1–7) (Ang-(1–7)). Assignments of the majority of the resonances were accomplished by COSY, TOCSY, and ROESY peak coordinates at 400 MHz and 600 MHz. Long-side-chain amino acid spin system identification was facilitated by long-range coherence transfer experiments (TOCSY). Problems with overlapped resonance signals were solved by analysis of heteronuclear 2D experiments (HSQC and HMBC). Nuclear Overhauser effects (NOE) results were used to probe peptide conformation. We show that the inclusion of the angiotensin-(1–7) tyrosine residue is favored in inclusion complexes with β-cyclodextrin. QM/MM simulations at the DFTB/UFF level confirm the experimental NMR findings and provide detailed structural information on these compounds in aqueous solution.

NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases. In this study, we present the solution structure of GIP in water determined by NMR spectroscopy. The calculated structure is characterized by the presence of an alpha-helical motif between residues Ser(11) and Gln(29). The helical conformation of GIP is further supported by CD spectroscopic studies. Six GIP-(1-42)Ala(1-7) analogues were synthesized by replacing individual N-terminal residues with alanine. Alanine scan studies of these N-terminal residues showed that the GIP-(1-42)Ala(6) was the only analogue to show insulin-secreting activity similar to that of the native GIP. However, when compared with glucose, its insulinotropic ability was reduced. For the first time, these NMR and modeling results contribute to the understanding of the structural requirements for the biological activity of GIP.

Biochemical Characterization of the Diaphanous Autoregulatory Interaction in the Formin Homology Protein FHOD1

Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site.

Probing Structure and Functional Dynamics of (Large) Proteins with Aromatic Rings: L-GFT-TROSY (4,3)D HCCH NMR Spectroscopy

NMR assignment of aromatic rings in proteins is a prerequisite for obtaining high-quality solution structures of proteins and for studying the dynamics and folding of their molecular cores. Here we present sensitive PFG-PEP L-GFT-(TROSY) (4,3)D HCCH NMR for identification of aromatic spin systems based on four-dimensional (4D) spectral information which can be rapidly obtained with high digital resolution. The G-matrix Fourier Transform (GFT) experiment relies on newly introduced longitudinal relaxation (L-)optimization for aromatic protons and is optimally suited for both sensitivity and sampling limited data collection, making it particularly attractive for NMR-based structural genomics. Applications are presented for 21 and 13 kDa proteins HR41 and MaR11, targets of the Northeast Structural Genomics Consortium for which data collection is, respectively, sensitivity and sampling limited. Complete assignment of aromatic rings enabled high-quality NMR structure determination, and nearly complete analysis of aromatic proton line widths allowed one to assess the flipping of most rings in HR41. Specifically, the ring of Tyr90 flips very slowly on the seconds time scale, thereby proving the absence of fast larger-amplitude motional modes which could allow the ring to flip. This indicates remarkable rigidity of the substructure in which the ring is embedded. Tyr90 is conserved among ubiquitin-conjugating enzymes E2, to which HR41 belongs, and is located in spatial proximity to the interface between E2 and ubiquitin protein ligase E3. Hence, the conformational rigidity and/or the slow motional mode probed by the ring might be of functional importance.

Probabilistic Identification of Spin Systems and their Assignments including Coil–Helix Inference as Output (PISTACHIO)

We present a novel automated strategy (PISTACHIO) for the probabilistic assignment of backbone and sidechain chemical shifts in proteins. The algorithm uses peak lists derived from various NMR experiments as input and provides as output ranked lists of assignments for all signals recognized in the input data as constituting spin systems. PISTACHIO was evaluated by comparing its performance with raw peak-picked data from 15 proteins ranging from 54 to 300 residues; the results were compared with those achieved by experts analyzing the same datasets by hand. As scored against the best available independent assignments for these proteins, the first-ranked PISTACHIO assignments were 80-100% correct for backbone signals and 75-95% correct for sidechain signals. The independent assignments benefited, in a number of cases, from structural data (e.g. from NOESY spectra) that were unavailable to PISTACHIO. Any number of datasets in any combination can serve as input. Thus PISTACHIO can be used as datasets are collected to ascertain the current extent of secure assignments, to identify residues with low assignment probability, and to suggest the types of additional data needed to remove ambiguities. The current implementation of PISTACHIO, which is available from a server on the Internet, supports input data from 15 standard double- and triple-resonance experiments. The software can readily accommodate additional types of experiments, including data from selectively labeled samples. The assignment probabilities can be carried forward and refined in subsequent steps leading to a structure. The performance of PISTACHIO showed no direct dependence on protein size, but correlated instead with data quality (completeness and signal-to-noise). PISTACHIO represents one component of a comprehensive probabilistic approach we are developing for the collection and analysis of protein NMR data.

Evidence for Domain-specific Recognition of SK and Kv Channels by MTX and HsTx1 Scorpion Toxins

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.

Solution structure of N-terminal segment of hepatitis B virus surface antigen Pre-S1

A synthetic peptide, the N-terminus of hepatitis B virus surface antigen Pre-S1, was studied by two-dimensional NMR techniques. A series of(1)H nuclear magnetic resonance experiments were used to complete the identification of spin systems and sequential assignments of this 28-residue peptide. 157 distance constraints and 55 dihedral angle constraints were obtained. 20 structures with the lowest target function were selected by the distance geometry program DIANA. Energy minimization and the following 100 ps time-averaged restrained molecular dynamics (TR-MD) simulation in aqueous solution were performed for each conformer. After TRMD simulation, three locally convergent regions corresponding to residues 22-31, 36-40, 41-46 were found. The averaged pairwise root-mean-square deviation (RMSD) of backbone atoms for them were (1.71+/-0.49)A, (0.76 +/-0.31)A, (1.05 +/- 0.52)A, respectively. Four reverse turns found in these regions, residues 22-25, 37-40, 41-44 and 43-46, correspond to several important antibody binding sites revealed in relevant immunological research.

Biophysical investigations on the myb-DNA system

The oncogene product c- myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions. The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c- myb and its cognate DNA target sequences. The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has revealed that the domain is largely helical in nature. Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases. Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different. Most of the conserved tryptophans are associated with the specific DNA binding site. Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein. In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.

Protein Heteronuclear NMR Assignments Using Mean-Field Simulated Annealing

A computational method for the assignment of the NMR spectra of larger (21 kDa) proteins using a set of six of the most sensitive heteronuclear multidimensional nuclear magnetic resonance experiments is described. Connectivity data obtained from HNCα, HN(CO)Cα, HN(Cα)Hα, and Hα(CαCO)NH and spin-system identification data obtained from CP-(H)CCH–TOCSY and CP-(H)C(CαCO)NH–TOCSY were used to perform sequence-specific assignments using a mean-field formalism and simulated annealing. This mean-field method reports the resonance assignments in a probabilistic fashion, displaying the certainty of assignments in an unambiguous and quantitative manner. This technique was applied to the NMR data of the 172-residue peptide-binding domain of theE. coliheat-shock protein, DnaK. The method is demonstrated to be robust to significant amounts of missing, spurious, noisy, extraneous, and erroneous data.

Use of deuterium labeling in NMR: overcoming a sizeable problem

In summary, the use of deuteration in combination with uniformly 13C- and 15N-labeling dramatically improves the quality of NMR spectra of larger proteins. These improvements allow the backbone and side-chain signals of larger proteins to be assigned and aids in the acquisition and analysis of NOE data. Using these methods, three-dimensional structures of proteins up to 30kDa have been determined [[[20]xStructure and ligand recognition of the phosphotyrosine binding domain of Shc. Zhou, M.-M...., and Fesik, S.W. Nature. 1995; 378: 584–592Crossref | PubMedSee all References, [31]xX-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Muchmore, S.W...., and Fesik, S.W. Nature. 1996; 381: 335–341Crossref | PubMedSee all References, [32]xStructure of Bcl-xL–Bak peptide complex reveals how regulators of apoptosis interact. Sattler, M...., and Fesik, S.W. Science. 1996; : in pressSee all References] SWF, unpublished data]. Further developments of NMR methods and variation of labeling techniques promise to push the molecular weight limit even further.

Three-dimensional 1H-TOCSY-relayed ct-[13C,1H]-HMQC for aromatic spin system identification in uniformly 13C-labeled proteins

Three-dimensional (1)H-TOCSY-relayed ct-[(13)C,(1)H]-HMQC is a novel experiment for aromatic spin system identification in uniformly (13)C-labeled proteins, which is implemented so that it correlates the chemical shift of a given aromatic proton with those of the directly attached carbon and all vicinal protons. The ct-HMQC scheme is used both for overlay of the indirect (1)H and (13)C chemical shift evolution periods and for the generation of (1)H-(1)H antiphase magnetization to accelerate the (1)H-TOCSY magnetization transfer at short mixing times. As an illustration, data recorded for the 18 kDa protein cyclophilin A are presented. Since transverse relaxation of (13)C-(1)H zero-quantum and double-quantum coherences is to first order insensitive to (13)C-(1)H heteronuclear dipolar relaxation, the new experiment should work also for proteins with molecular weights above 20 kDa.