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.     

An assignment of intrinsically disordered regions of proteins based on NMR structures

Intrinsically disordered proteins (IDPs) do not adopt stable three-dimensional structures in physiological conditions, yet these proteins play crucial roles in biological phenomena. In most cases, intrinsic disorder manifests itself in segments or domains of an IDP, called intrinsically disordered regions (IDRs), but fully disordered IDPs also exist. Although IDRs can be detected as missing residues in protein structures determined by X-ray crystallography, no protocol has been developed to identify IDRs from structures obtained by Nuclear Magnetic Resonance (NMR). Here, we propose a computational method to assign IDRs based on NMR structures. We compared missing residues of X-ray structures with residue-wise deviations of NMR structures for identical proteins, and derived a threshold deviation that gives the best correlation of ordered and disordered regions of both structures. The obtained threshold of 3.2 Å was applied to proteins whose structures were only determined by NMR, and the resulting IDRs were analyzed and compared to those of X-ray structures with no NMR counterpart in terms of sequence length, IDR fraction, protein function, cellular location, and amino acid composition, all of which suggest distinct characteristics. The structural knowledge of IDPs is still inadequate compared with that of structured proteins. Our method can collect and utilize IDRs from structures determined by NMR, potentially enhancing the understanding of IDPs.     

HA-detected experiments for the backbone assignment of intrinsically disordered proteins

We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual ¹⁵N(i)-¹³C′(i)-H^α(i) (or ¹⁵N(i)-¹³C^α(i)-H^α(i)) and sequential ¹⁵N(i + 1)-¹³C′(i)-H^α(i) (or ¹⁵N(i + 1)-¹³C^α(i)-H^α(i)) correlation experiments together with efficient use of chemical shifts of ¹⁵N and ¹³C′ nuclei, which show smaller dependence on residue type. We have tested the proposed protocol on two proteins, small globular 56-residue GB1, and highly disordered, proline-rich 47-residue fifth repeat of EspF_U. Using the proposed approach, we were able to assign 90% of ¹H^α, ¹³C^α, ¹³C′, ¹⁵N chemical shifts in EspF_U. We reckon that the HA-detection based strategy will be very useful in the assignment of natively unfolded proline-rich proteins or polypeptide chains.     

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.     

BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins

The characterization of the conformational properties of intrinsically disordered proteins (IDPs), and their interaction modes with physiological partners has recently become a major research topic for understanding biological function on the molecular level. Although multidimensional NMR spectroscopy is the technique of choice for the study of IDPs at atomic resolution, the intrinsically low resolution, and the large peak intensity variations often observed in NMR spectra of IDPs call for resolution- and sensitivity-optimized pulse schemes. We present here a set of amide proton-detected 3D BEST-TROSY correlation experiments that yield the required sensitivity and spectral resolution for time-efficient sequential resonance assignment of large IDPs. In addition, we introduce two proline-edited 2D experiments that allow unambiguous identification of residues adjacent to proline that is one of the most abundant amino acids in IDPs. The performance of these experiments, and the advantages of BEST-TROSY pulse schemes are discussed and illustrated for two IDPs of similar length (~270 residues) but with different conformational sampling properties.     

New 13C-detected experiments for the assignment of intrinsically disordered proteins

NMR assignment of intrinsically disordered proteins (IDPs) by conventional HN-detected methods is hampered by the small dispersion of the amide protons chemical shifts and exchange broadening of amide proton signals. Therefore several alternative assignment strategies have been proposed in the last years. Attempting to seize that dispersion of ¹³C′ and ¹⁵N chemical shifts holds even in IDPs, we recently proposed two ¹³C-detected experiments to directly correlate the chemical shifts of two consecutive ¹³C′–¹⁵N groups in proteins, i.e. without mediation of other nuclei. Main drawback of these experiments is the interruption of the connection at prolines. Here we present new ¹³C-detected experiments to correlate consecutive ¹³C′–¹⁵N groups in IDPs, hacacoNcaNCO and hacaCOncaNCO, that overcome this limitation. Moreover, the experiments provide recognition of glycine residues, thereby facilitating the assignment process.     

5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of ¹³C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.     

ncIDP-assign: a SPARKY extension for the effective NMR assignment of intrinsically disordered proteins

We describe here the ncIDP-assign extension for the popular NMR assignment program SPARKY, which aids in the sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The assignment plugin greatly facilitates the effective matching of a set of connected resonances to the correct position in the sequence by making use of IDP random coil chemical shifts. AVAILABILITY: The ncIDP-assign extension is available at http://www.protein-nmr.org/.     

A complete set of novel 2D correlation NMR experiments based on heteronuclear J-cross polarization

A suite of multiple-purpose sensitivity-enhanced 2D correlation NMR experiments based on heteronuclear J-cross polarization (HCP) techniques are introduced for isotropic liquid samples. Several pulse sequences using an adaptable heteronuclear TOCSY mixing building block are proposed for different types of effective coherence-order-selective (COS) heteronuclear coherence-transfer mechanisms. They are based on the anisotropic behaviour of the involved HCP process that is easily described and analysed in terms of cartesian product-operator formalism. A number of different versions are given for in-phase to in-phase (II-COS: S (-) \\ to I (-)), in-phase to anti-phase (IA-COS: S (-) \\ to 2 I (-) S (z)), in-phase to spin-state-selective (IS(3)-COS: S (-) \\ to 2 I (-) S (alpha /beta)), anti-phase to in-phase (AI-COS: 2 I (z) S (-) \\ to I (-)), anti-phase to anti-phase (AA-COS: 2 I (z) S (-) \\ to 2 I (-) S (z)), anti-phase to spin-state-selective (AS(3)-COS: 2 I (z) S (-) \\ to 2 I (-) S (alpha /beta)) and spin-state-selective to spin-state-selective (S(3)S(3)-COS: 2 I (alpha /beta) S (-) \\ to 2 I (-) S (alpha /beta )) coherence transfers. The combination of the echo/anti-echo approach, heteronuclear gradient echoes and the preservation of equivalent pathways (PEP) methodology affords a general approach to obtain sensitivity-enhanced pure-absorption 2D spectra that can be used as interesting alternatives to conventional pulse-interrupted free-precession INEPT-based pulse schemes, such as HSQC-type and TROSY-type experiments.     

High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using δ subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences.     

Sequential resonance assignment of medium-sized15 N/13C-labeled proteins with projected 4D triple resonance NMR experiments

We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of ¹⁵N/¹³C-labeled proteins. A 3D ^{/ /}(CO)NHN experiment is recorded either in conjunction with 3D HNN< > or with the newly presented 3D HNN scheme. The first combination yields sequential assignments based on the measurement of¹³ C chemical shifts and provides a complete ¹H, ¹³C and ¹⁵N resonance assignment of polypeptide backbone and CH_n moieties. When employing the second combination, ¹³C=O chemical shifts are not measured, but the sequential assignment relies on both ¹³C and¹ H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized¹⁵ N/¹³C doubly labeled proteins.     

Improved 4D NMR experiments for the assignment of backbone nuclei in13C/15N labelled proteins

Summary We recently proposed a novel 4D NMR strategy for the assignment of backbone nuclei in¹³C/¹⁵N-labelled proteins (Boucher et al., 1992). Intra-residue (and many sequential) assignments are obtained from a HCANNH experiment, whereas sequential assignments are based on a complementary HCA(CO)NNH experiment. We present here new constant time 4D HCANNH, HCA(CO)NNH and HNCAHA experiments that are more sensitive. Some of the data were presented at the 33rd ENC held at Asilomar, California, U.S.A., in April 1992.     

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 reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins

Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue—HN, N, CA and CO chemical shifts—from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i ? 1) amide ¹H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135–147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.     

HN(CA)N and HN(COCA)N experiments for assignment of large disordered proteins

Two new 3D HN-based experiments are proposed for backbone assignment of large disordered proteins. The spectra obtained with the new pulse schemes are free of redundant diagonal peaks (H_iN_i–N_i) and provide sequential correlations (H_iN_i–N_i+1 and H_iN_i–N_i?1) not only between adjacent non-proline residues but also between non-proline and proline residues. The experiments have been demonstrated on an intrinsically disordered protein with 306 amino acids including 64 proline residues. Using the two experiments, we obtained nearly complete assignments of backbone amides and proline ¹⁵N spins except for 4 proline and 4 non-proline residues.     

Resonance assignment of the NMR spectra of disordered proteins using a multi-objective non-dominated sorting genetic algorithm

A multi-objective genetic algorithm is introduced to predict the assignment of protein solid-state NMR (SSNMR) spectra with partial resonance overlap and missing peaks due to broad linewidths, molecular motion, and low sensitivity. This non-dominated sorting genetic algorithm II (NSGA-II) aims to identify all possible assignments that are consistent with the spectra and to compare the relative merit of these assignments. Our approach is modeled after the recently introduced Monte-Carlo simulated-annealing (MC/SA) protocol, with the key difference that NSGA-II simultaneously optimizes multiple assignment objectives instead of searching for possible assignments based on a single composite score. The multiple objectives include maximizing the number of consistently assigned peaks between multiple spectra (“good connections”), maximizing the number of used peaks, minimizing the number of inconsistently assigned peaks between spectra (“bad connections”), and minimizing the number of assigned peaks that have no matching peaks in the other spectra (“edges”). Using six SSNMR protein chemical shift datasets with varying levels of imperfection that was introduced by peak deletion, random chemical shift changes, and manual peak picking of spectra with moderately broad linewidths, we show that the NSGA-II algorithm produces a large number of valid and good assignments rapidly. For high-quality chemical shift peak lists, NSGA-II and MC/SA perform similarly well. However, when the peak lists contain many missing peaks that are uncorrelated between different spectra and have chemical shift deviations between spectra, the modified NSGA-II produces a larger number of valid solutions than MC/SA, and is more effective at distinguishing good from mediocre assignments by avoiding the hazard of suboptimal weighting factors for the various objectives. These two advantages, namely diversity and better evaluation, lead to a higher probability of predicting the correct assignment for a larger number of residues. On the other hand, when there are multiple equally good assignments that are significantly different from each other, the modified NSGA-II is less efficient than MC/SA in finding all the solutions. This problem is solved by a combined NSGA-II/MC algorithm, which appears to have the advantages of both NSGA-II and MC/SA. This combination algorithm is robust for the three most difficult chemical shift datasets examined here and is expected to give the highest-quality de novo assignment of challenging protein NMR spectra.     

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.     

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.