Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving ¹³C and ¹⁵N spins have been developed for this purpose, ¹H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular ¹H-¹³C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved ¹H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR, simplifies the ¹H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced ¹H chemical shift resolution allows the assignment of overlapping H and H resonances in Ser, identifies the ¹H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed.     

Prospects for resonance assignments in multidimensional solid-state NMR spectra of uniformly labeled proteins

Summary The feasibility of assigning the backbone ¹⁵N and ¹³C NMR chemical shifts in multidimensional magic angle spinning NMR spectra of uniformly isotopically labeled proteins and peptides in unoriented solid samples is assessed by means of numerical simulations. The goal of these simulations is to examine how the upper limit on the size of a peptide for which unique assignments can be made depends on the spectral resolution, i.e., the NMR line widths. Sets of simulated three-dimensional chemical shift correlation spectra for artificial peptides of varying length are constructed from published liquid-state NMR chemical shift data for ubiquitin, a well-characterized soluble protein. Resonance assignments consistent with these spectra to within the assumed spectral resolution are found by a numerical search algorithm. The dependence of the number of consistent assignments on the assumed spectral resolution and on the length of the peptide is reported. If only three-dimensional chemical shift correlation data for backbone ¹⁵N and ¹³C nuclei are used, and no residue-specific chemical shift information, information from amino acid side-chain signals, and proton chemical shift information are available, a spectral resolution of 1 ppm or less is generally required for a unique assignment of backbone chemical shifts for a peptide of 30 amino acid residues.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

PROSHIFT: protein chemical shift prediction using artificial neural networks

The importance of protein chemical shift values for the determination of three-dimensional protein structure has increased in recent years because of the large databases of protein structures with assigned chemical shift data. These databases have allowed the investigation of the quantitative relationship between chemical shift values obtained by liquid state NMR spectroscopy and the three-dimensional structure of proteins. A neural network was trained to predict the ¹H, ¹³C, and ¹⁵N of proteins using their three-dimensional structure as well as experimental conditions as input parameters. It achieves root mean square deviations of 0.3 ppm for hydrogen, 1.3 ppm for carbon, and 2.6 ppm for nitrogen chemical shifts. The model reflects important influences of the covalent structure as well as of the conformation not only for backbone atoms (as, e.g., the chemical shift index) but also for side-chain nuclei. For protein models with a RMSD smaller than 5 Å a correlation of the RMSD and the r.m.s. deviation between the predicted and the experimental chemical shift is obtained. Thus the method has the potential to not only support the assignment process of proteins but also help with the validation and the refinement of three-dimensional structural proposals. It is freely available for academic users at the PROSHIFT server: www.jens-meiler.de/proshift.html     

A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles

Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the ??shotgun?? approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the ¹⁵N chemical shift and ¹⁵N?C¹H dipolar couplings, as well as spin-correlations between the (i, i?±?1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an ¹⁵N?C¹⁵N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled ??seed?? spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B ₁ fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.     

Influence of the completeness of chemical shift assignments on NMR structures obtained with automated NOE assignment

Reliable automated NOE assignment and structure calculation on the basis of a largely complete, assigned input chemical shift list and a list of unassigned NOESY cross peaks has recently become feasible for routine NMR protein structure calculation and has been shown to yield results that are equivalent to those of the conventional, manual approach. However, these algorithms rely on the availability of a virtually complete list of the chemical shifts. This paper investigates the influence of incomplete chemical shift assignments on the reliability of NMR structures obtained with automated NOESY cross peak assignment. The program CYANA was used for combined automated NOESY assignment with the CANDID algorithm and structure calculations with torsion angle dynamics at various degrees of completeness of the chemical shift assignment which was simulated by random omission of entries in the experimental ¹H chemical shift lists that had been used for the earlier, conventional structure determinations of two proteins. Sets of structure calculations were performed choosing the omitted chemical shifts randomly among all assigned hydrogen atoms, or among aromatic hydrogen atoms. For comparison, automated NOESY assignment and structure calculations were performed with the complete experimental chemical shift but under random omission of NOESY cross peaks. When heteronuclear-resolved three-dimensional NOESY spectra are available the current CANDID algorithm yields in the absence of up to about 10% of the experimental ¹H chemical shifts reliable NOE assignments and three-dimensional structures that deviate by less than 2 Å from the reference structure obtained using all experimental chemical shift assignments. In contrast, the algorithm can accommodate the omission of up to 50% of the cross peaks in heteronuclear- resolved NOESY spectra without producing structures with a RMSD of more than 2 Å to the reference structure. When only homonuclear NOESY spectra are available, the algorithm is slightly more susceptible to missing data and can tolerate the absence of up to about 7% of the experimental ¹H chemical shifts or of up to 30% of the NOESY peaks.Abbreviations: BmPBP^A – Bombyx mori pheromone binding protein form A; CYANA – combined assignment and dynamics algorithm for NMR applications; NMR – nuclear magnetic resonance; NOE – nuclear Overhauser effect; NOESY – NOE spectroscopy; RMSD – root-mean-square deviation; WmKT – Williopsis mrakii killer toxin     

Automated and assisted RNA resonance assignment using NMR chemical shift statistics

The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and ¹H and ¹³C chemical shifts. Statistics of resonances from regularly Watson–Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1′/C1′ chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding ¹³C resonances, which provides an important step toward automated structure determination of RNAs.     

Automatic <Superscript>13</Superscript>C chemical shift reference correction for unassigned protein NMR spectra

Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR, including protein structure determination and analysis of protein dynamics. To solve this problem, we constructed a Bayesian probabilistic framework that circumvents the limitations of previous reference correction methods that required protein resonance assignment and/or three-dimensional protein structure. Our algorithm named Bayesian Model Optimized Reference Correction (BaMORC) can detect and correct ¹³C chemical shift referencing errors before the protein resonance assignment step of analysis and without three-dimensional structure. By combining the BaMORC methodology with a new intra-peaklist grouping algorithm, we created a combined method called Unassigned BaMORC that utilizes only unassigned experimental peak lists and the amino acid sequence. Unassigned BaMORC kept all experimental three-dimensional HN(CO)CACB-type peak lists tested within ±?0.4 ppm of the correct ¹³C reference value. On a much larger unassigned chemical shift test set, the base method kept ¹³C chemical shift referencing errors to within ±?0.45 ppm at a 90% confidence interval. With chemical shift assignments, Assigned BaMORC can detect and correct ¹³C chemical shift referencing errors to within ±?0.22 at a 90% confidence interval. Therefore, Unassigned BaMORC can correct ¹³C chemical shift referencing errors when it will have the most impact, right before protein resonance assignment and other downstream analyses are started. After assignment, chemical shift reference correction can be further refined with Assigned BaMORC. These new methods will allow non-NMR experts to detect and correct ¹³C referencing error at critical early data analysis steps, lowering the bar of NMR expertise required for effective protein NMR analysis.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy

The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (153 residues, Mr = 17,400) has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. We show that the problems of amide NH and C alpha H chemical shift degeneracy that are prevalent for proteins of this size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1-interleukin 1 beta. The complete list of 15N and 1H assignments is given for all the amide NH and C alpha H resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1 beta.     

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.     

13C NMR spectra of some naturally occurring binaphthoquinones and related compounds

¹³C NMR chemical shift assignments have been shown to be diagnostic for the establishment of the dimeric linkage of some naturally occurring binaphthoquinones. The unsymmetric ¹³C and ¹H spin-spin coupled pattern observed in the ¹H coupled ¹³C NMR spectrum of plumbagin for C-6 has also been noticed earlier with the related compound juglone. The nature of these effects has been substantiated for the first time using benzene induced solvent shifts and D₂O exchange. ¹³C chemical shift assignments of plumbagin reported earlier for C-6 and C-8 have been revised.     

Automated assignment of NMR chemical shifts using peak-particle dynamics simulation with the DYNASSIGN algorithm

A new algorithm, DYNASSIGN, for the automated assignment of NMR chemical shift resonances was developed in which expected cross peaks in multidimensional NMR spectra are represented by peak-particles and assignment restraints are translated into a potential energy function. Molecular dynamics simulation techniques are used to calculate a trajectory of the system of peak-particles subjected to the potential function in order to find energetically optimal configurations that correspond to correct assignments. Peak-particle dynamics-based simulated annealing was combined with the Hungarian algorithm for local optimization, and a residue-based score was introduced to distinguish between reliable assignments and “unassigned” resonances for which no reliable assignment can be established. The DYNASSIGN algorithm was implemented in the program CYANA and tested with data sets obtained from the experimental NMR data of nine small proteins. With a set of 10 commonly used NMR spectra, on average 82.5% of all backbone and side-chain ¹H, ¹³C and ¹⁵N resonances could be assigned with an average error rate of 3.5%.     

Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS

Novel algorithms are presented for automated NOESY peak picking and NOE signal identification in homonuclear 2D and heteronuclear-resolved 3D [¹H,¹H]-NOESY spectra during de novoprotein structure determination by NMR, which have been implemented in the new software ATNOS (automated NOESY peak picking). The input for ATNOS consists of the amino acid sequence of the protein, chemical shift lists from the sequence-specific resonance assignment, and one or several 2D or 3D NOESY spectra. In the present implementation, ATNOS performs multiple cycles of NOE peak identification in concert with automated NOE assignment with the software CANDID and protein structure calculation with the program DYANA. In the second and subsequent cycles, the intermediate protein structures are used as an additional guide for the interpretation of the NOESY spectra. By incorporating the analysis of the raw NMR data into the process of automated de novoprotein NMR structure determination, ATNOS enables direct feedback between the protein structure, the NOE assignments and the experimental NOESY spectra. The main elements of the algorithms for NOESY spectral analysis are techniques for local baseline correction and evaluation of local noise level amplitudes, automated determination of spectrum-specific threshold parameters, the use of symmetry relations, and the inclusion of the chemical shift information and the intermediate protein structures in the process of distinguishing between NOE peaks and artifacts. The ATNOS procedure has been validated with experimental NMR data sets of three proteins, for which high-quality NMR structures had previously been obtained by interactive interpretation of the NOESY spectra. The ATNOS-based structures coincide closely with those obtained with interactive peak picking. Overall, we present the algorithms used in this paper as a further important step towards objective and efficient de novoprotein structure determination by NMR.     

<Superscript>13</Superscript>C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides

We present here a set of ¹³C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose ¹³C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of ¹³C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, ¹³C-direct detected NMR methods constitute useful complements to the conventional ¹H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.     

Efficient sequential assignments in proteins with reduced dimensionality 3D HN(CA)NH

We present reduced dimensionality (RD) 3D HN(CA)NH for efficient sequential assignment in proteins. The experiment correlates the ¹⁵N and ¹H chemical shift of a residue (‘i’) with those of its immediate N-terminal (i − 1) and C-terminal (i + 1) neighbors and provides four-dimensional chemical shift correlations rapidly with high resolution. An assignment strategy is presented which combines the correlations observed in this experiment with amino acid type information obtained from 3D CBCA(CO)NH. By classifying the 20 amino acid types into seven distinct categories based on ¹³C^β chemical shifts, it is observed that a stretch of five sequentially connected residues is sufficient to map uniquely on to the polypeptide for sequence specific resonance assignments. This method is exemplified by application to three different systems: maltose binding protein (42 kDa), intrinsically disordered domain of insulin-like growth factor binding protein-2 and Ubiquitin. Fast data acquisition is demonstrated using longitudinal ¹H relaxation optimization. Overall, 3D HN(CA)NH is a powerful tool for high throughput resonance assignment, in particular for unfolded or intrinsically disordered polypeptides.     

NMR with 13C, 15N-doubly-labeled DNA: The shape Antennapedia homeodomain complex with a 14-mer DNA duplex

Nearly complete ¹H, ¹³C and¹⁵ N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly ¹⁵N-labeled Antennapedia homeodomain. The DNA was either fully ¹³C,¹⁵N-labeled or contained uniformly ¹³C, ¹⁵N-labeled nucleotides only at those positions which form the protein–DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain–DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via¹ H–¹H NOEs observed in 3D¹³ C-resolved NOESY; and (iii) assignment of the imino and amino groups via ¹H–¹H NOEs and¹⁵ N–¹H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein–DNA complex was greatly facilitated by the fact that ¹H signals of the protein were filtered out in ¹³C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding ¹³C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.