Robust structure-based resonance assignment for functional protein studies by NMR

High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly H ^N –H ^N NOEs networks, as well as ¹ H–¹⁵ N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: .     

BSH-CP based 3D solid-state NMR experiments for protein resonance assignment

We have recently presented band-selective homonuclear cross-polarization (BSH-CP) as an efficient method for CO–CA transfer in deuterated as well as protonated solid proteins. Here we show how the BSH-CP CO–CA transfer block can be incorporated in a set of three-dimensional (3D) solid-state NMR (ssNMR) pulse schemes tailored for resonance assignment of proteins at high static magnetic fields and moderate magic-angle spinning rates. Due to the achieved excellent transfer efficiency of 33 % for BSH-CP, a complete set of 3D spectra needed for unambiguous resonance assignment could be rapidly recorded within 1 week for the model protein ubiquitin. Thus we expect that BSH-CP could replace the typically used CO–CA transfer schemes in well-established 3D ssNMR approaches for resonance assignment of solid biomolecules.     

Robust refocusing of 13C magnetization in multidimensional NMR experiments by adiabatic fast passage pulses

We show that adiabatic fast passage (AFP) pulses are robust refocusing elements of transverse ¹³C magnetization in multidimensional NMR experiments. A pair of identical AFP pulses can refocus selected parts or a complete ¹³ C chemical shift range in ¹³C spectra. In the constant time ¹³C-¹H HSQC, replacement of attenuated rectangular pulses by selective AFP pulses results in a sensitivity enhancement of up to a factor of 1.8. In the 3D CBCA(CO)NH the signal-to-noise ratio is increased by a factor of up to 1.6.     

Sensitivity enhancement in NMR of macromolecules by application of optimal control theory

NMR of macromolecules is limited by large transverse relaxation rates. In practice, this results in low efficiency of coherence transfer steps in multidimensional NMR experiments, leading to poor sensitivity and long acquisition times. The efficiency of coherence transfer can be maximized by design of relaxation optimized pulse sequences using tools from optimal control theory. In this paper, we demonstrate that this approach can be adopted for studies of large biological systems, such as the 800 kDa chaperone GroEL. For this system, the ¹H–¹⁵N coherence transfer module presented here yields an average sensitivity enhancement of 20–25% for cross-correlated relaxation induced polarization transfer (CRIPT) experiments.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-3592-0     

High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins

We present three novel exclusively heteronuclear 5D ¹³C direct-detected NMR experiments, namely (H^N-flipN)CONCACON, (HCA)CONCACON and (H)CACON(CA)CON, designed for easy sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The experiments proposed have been optimized to overcome the drawbacks which may dramatically complicate the characterization of IDPs by NMR, namely the small dispersion of chemical shifts and the fast exchange of the amide protons with the solvent. A fast and reliable automatic assignment of α-synuclein chemical shifts was obtained with the Tool for SMFT-based Assignment of Resonances (TSAR) program based on the information provided by these experiments.     

A novel computational approach to integrate NMR spectroscopy and capillary electrophoresis for structure assignment of heparin and heparan sulfate oligosaccharides

Heparin and heparan sulfate (HS) glycosaminoglycans (GAGs) are cell surface polysaccharides that bind to a multitude of signaling molecules, enzymes, and pathogens and modulate critical biological processes ranging from cell growth and development to anticoagulation and viral invasion. Heparin has been widely used as an anticoagulant in a variety of clinical applications for several decades. The heterogeneity and complexity of HS GAGs pose significant challenges to their purification and characterization of structure-function relationships. Nuclear magnetic resonance (NMR) spectroscopy is a promising tool that provides abundant sequence and structure information for characterization of HS GAGs. However, complex NMR spectra and low sensitivity often make analysis of HS GAGs a daunting task. We report the development of a novel methodology that incorporates distinct linkage information between adjacent monosaccharides obtained from NMR and capillary electrophoresis (CE) data using a property encoded nomenclature (PEN) computational framework to facilitate a rapid and unbiased procedure for sequencing HS GAG oligosaccharides. We demonstrate that the integration of NMR and CE data sets with the help of the PEN framework dramatically reduces the number of experimental constraints required to arrive at an HS GAG oligosaccharide sequence.     

High field NMR study of antibiotic peptides: 1H assignment of trichorzianine A1 spectra by 2D experiments

A new antibiotic peptide, trichorzianine A1, was isolated from a culture of Trichoderma harzianum. It contains 19 residues, the N terminus is blocked by an acetyl group and the C terminus is tryptophanol. As a first part of the structural study of this new peptide, we here present the analysis of the 1H NMR spectrum accomplished by 2 DJ resolved and spin echo correlated spectroscopy.     

NMR assignment of human chemerin, a novel chemoattractant

Chemerin is a potent chemoattractant for cells expressing the GPCR CMKLR1, and is thought to play important roles in cell migration and recruitment to sites of tissue damage and inflammation. Here we report the NMR assignments of the 15.6 kDa active form of uniformly ¹⁵N, ¹³C labeled chemerin.     

A random graph approach to NMR sequential assignment.

Nuclear magnetic resonance (NMR) spectroscopy allows scientists to study protein structure, dynamics and interactions in solution. A necessary first step for such applications is determining the resonance assignment, mapping spectral data to atoms and residues in the primary sequence. Automated resonance assignment algorithms rely on information regarding connectivity (e.g., through-bond atomic interactions) and amino acid type, typically using the former to determine strings of connected residues and the latter to map those strings to positions in the primary sequence. Significant ambiguity exists in both connectivity and amino acid type information. This paper focuses on the information content available in connectivity alone and develops a novel random-graph theoretic framework and algorithm for connectivity-driven NMR sequential assignment. Our random graph model captures the structure of chemical shift degeneracy, a key source of connectivity ambiguity. We then give a simple and natural randomized algorithm for finding optimal assignments as sets of connected fragments in NMR graphs. The algorithm naturally and efficiently reuses substrings while exploring connectivity choices; it overcomes local ambiguity by enforcing global consistency of all choices. By analyzing our algorithm under our random graph model, we show that it can provably tolerate relatively large ambiguity while still giving expected optimal performance in polynomial time. We present results from practical applications of the algorithm to experimental datasets from a variety of proteins and experimental set-ups. We demonstrate that our approach is able to overcome significant noise and local ambiguity in identifying significant fragments of sequential assignments.     

2D selective-TOCSY-DQFCOSY and HSQC-TOCSY NMR experiments for assignment of a homogeneous asparagine-linked triantennary complex type undecasaccharide

The assignment of ¹H and ¹³C NMR signals of a complex type triantennary asialooligosaccharide was examined using 2D selective-TOCSY–DQFCOSY and HSQC–TOCSY experiments. The 2D selective-TOCSY–DQFCOSY experiment exhibits a 2D DQFCOSY spectrum of an individual monosaccharide in the undecasaccharide, although the NMR signals of several monosaccharides in the triantennary undecasaccharide are heavily overlapped. Selective excitation of each anomeric proton signal and subsequent TOCSY experiment afforded transverse magnetization corresponding to all of the proton signals of the monosaccharide. This magnetization was then developed with the corresponding DQFCOSY pulse sequence to afford the DQFCOSY spectrum of the individual monosugars. In this case, four GlcNAc-b, -e, -j, and -h residues were excited as a mixture. In order to assign ¹³C signals, a conventional 2D HSQC–TOCSY spectrum was examined and compared with an unambiguous assignment of 2D selective-TOCSY–DQFCOSY thus obtained. This systematic analysis made it possible to obtain an assignment of the ¹H and ¹³C NMR signals of the triantennary undecasaccharide. In addition, these experiments also revealed all of the glycosyl positions in the triantennary undecasaccharide.     

Monitoring anthracycline binding to nucleotides by two dimensional cosy NMR experiments.

The nucleotide binding ability of the novel anthracycline drug, 3-fluoro-4-demethoxydaunomycin, has been studied by two dimensional 1H NMR correlated spectroscopy (COSY). In the COSY spectrum of the nucleotide mini-helix d(CTGCAG)2 cross-peaks are observed from the spin-coupled H6 and H5 protons of the cytidine bases. Additional cytidine H6/H5 cross-peaks are observed in the COSY spectrum of the anthracycline-d(CTGCAG)2 complex. These additional cytidine cross-peaks enable the identification of the anthracycline binding sites and the determination of the relative kinetic stability of the bound drug at each binding site.     

Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments

A fast, robust and reliable strategy for automated sequential resonance assignment for uniformly [¹³C, ¹⁵N]-labeled RNA via its phosphodiester backbone is presented. It is based on a series of high-dimensional through-bond APSY experiments: a 5D HCP-CCH COSY, a 4D H1′C1′CH TOCSY for ribose resonances, a 5D HCNCH for ribose-to-base connection, a 4D H6C6C5H5 TOCSY for pyrimidine resonances, and a 4D H8C8(C)C2H2 TOCSY for adenine resonances. The utilized pulse sequences are partially novel, and optimized to enable long evolution times in all dimensions. The highly precise APSY peak lists derived with these experiments could be used directly for reliable automated resonance assignment with the FLYA algorithm. This approach resulted in 98 % assignment completeness for all ¹³C–¹H, ¹⁵N1/9 and ³¹P resonances of a stem-loop with 14 nucleotides.     

Improved NMR experiments with 13C-isotropic mixing for assignment of aromatic and aliphatic side chains in labeled proteins

Three improved ¹³C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(C^aliC^aro)H-TOCSY, is an HCCH-TOCSY in which all CH_n moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the ¹³C-detected C^all-TOCSY, magnetization transfer comprises all carbons—aliphatic, aromatic as well as the carbonyl carbons—making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct ¹³C–¹³C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa.     

A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

The determination of the three-dimensional structure of a protein or the study of protein–ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p. Dedicated to Rüdiger Winter ( 06.04.2004)     

Integration of splicing, transport and translation to achieve mRNA quality control by the nonsense-mediated decay pathway

When pre-mRNAs are spliced, a multi-component complex is deposited onto them, close to the sites of intron removal. New findings suggest that these exon-exon junction complexes and the complexes that bind mRNA caps are key effectors of the fate of spliced mRNAs and may regulate whether mRNAs containing premature stop codons are degraded.     

Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments

A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.