Search

Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations. Understanding the function of these complexes requires the characterization of the entire accessible conformation space and its modulation by interaction partners. Long-range (1.8-10 nm) distance restraints obtained by EPR spectroscopy in the form of probability distributions are ideally suited for this task as not only the mean distance but also information about the conformation dynamics is encoded in the experimental data. Here we describe a computational method that we have developed based on well-established structure determination software using NMR restraints to calculate the accessible conformation space using PELDOR/DEER data., (© 2023. The Author(s).)

Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15 N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative "Stablelabel" containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in "Stablelabel" lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin., (© 2023. The Author(s).)

Recent advances in the field of protein structure determination using liquid-state NMR enable the elucidation of multi-state protein conformations that can provide insight into correlated and non-correlated protein dynamics at atomic resolution. So far, NMR-derived multi-state structures were typically evaluated by means of visual inspection of structure superpositions, target function values that quantify the violation of experimented restraints and root-mean-square deviations that quantify similarity between conformers. As an alternative or complementary approach, we present here the use of a recently introduced structural correlation measure, PDBcor, that quantifies the clustering of protein states as an additional measure for multi-state protein structure analysis. It can be used for various assays including the validation of experimental distance restraints, optimization of the number of protein states, estimation of protein state populations, identification of key distance restraints, NOE network analysis and semiquantitative analysis of the protein correlation network. We present applications for the final quality analysis stages of typical multi-state protein structure calculations., (© 2022. The Author(s).)

NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM 4 A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing., (© 2021. The Author(s).)

Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and long-term, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of "poised fragments" with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process.

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13 C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, C α , C β , C') resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between β-strands performing an inward movement and that this movement also affects the entire β-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.

Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927-7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1 H, 31 P-correlation experiments as well as a BEST-HNP experiment exploiting 3h J N,P rather than 2h J H,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~ 100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2'-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.

Recently, 15 N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15 N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three 15 N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15 N-detected BEST-TROSY experiment, which proves to be the most sensitive 15 N-detected H-N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the 15 N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1 H- than in the 15 N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15 N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.

Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1β (Barthelmes et al. in J Am Chem Soc 133:808-819, 2011). Here, we investigate the differences in fast dynamics of selected loop-LBT interleukin-1β constructs by measuring 15 N nuclear spin relaxation experiments. We show that the loop-LBT does not significantly alter the dynamic motions of the host protein in the sub-τ c -timescale and that the loop-LBT adopts a rigid conformation with significantly reduced dynamics compared to the terminally attached encodable LBT leading to increased paramagnetic alignment strength. We further analyze residual dipolar couplings (RDCs) obtained by loop-LBTs and additional liquid crystalline media to assess the applicability of the loop-LBT approach for RDC-based methods to determine structure and dynamics of proteins, including supra-τc dynamics. Using orthogonalized linear combinations (OLCs) of RDCs and Saupe matrices, we show that the combined use of encodable LBTs and external alignment media yields up to five linear independent alignments.

Paramagnetic relaxation enhancement (PRE) is a versatile tool for NMR spectroscopic structural and kinetic studies in biological macromolecules. Here, we compare the quality of PRE data derived from two spin labels with markedly different dynamic properties for large RNAs using the I-A riboswitch aptamer domain (78 nt) from Mesoplamsa florum as model system. We designed two I-A aptamer constructs that were spin-labeled by noncovalent hybridization of short spin-labeled oligomer fragments. As an example of a flexible spin label, Ureido U-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Çm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromatic 13 C bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (~78 nt). PRE restraints derived from both imino and aromatic protons yielded similar data quality, and hence can both be reliably used for PRE determination. For NMR, the data quality derived from the rigid spin label Çm is slightly better than the data quality for the flexible Ureido TEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).

The automated identification of signals in multidimensional NMR spectra is a challenging task, complicated by signal overlap, noise, and spectral artifacts, for which no universally accepted method is available. Here, we present a new peak picking algorithm, CYPICK, that follows, as far as possible, the manual approach taken by a spectroscopist who analyzes peak patterns in contour plots of the spectrum, but is fully automated. Human visual inspection is replaced by the evaluation of geometric criteria applied to contour lines, such as local extremality, approximate circularity (after appropriate scaling of the spectrum axes), and convexity. The performance of CYPICK was evaluated for a variety of spectra from different proteins by systematic comparison with peak lists obtained by other, manual or automated, peak picking methods, as well as by analyzing the results of automated chemical shift assignment and structure calculation based on input peak lists from CYPICK. The results show that CYPICK yielded peak lists that compare in most cases favorably to those obtained by other automated peak pickers with respect to the criteria of finding a maximal number of real signals, a minimal number of artifact peaks, and maximal correctness of the chemical shift assignments and the three-dimensional structure obtained by fully automated assignment and structure calculation.

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.

In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus (19)F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5'-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the (19)F isotope. The thermal stability of the (19)F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a (1)H,(15)N-HSQC allow the identification of Watson-Crick base paired uridine signals and the (19)F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of (19)F-labeling even for sizeable RNAs in the range of 70 nucleotides.

We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1β (IL1β) and measured (1)H, (15)N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm(3+)-loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1β-S2R2 and the respective single-loop-LBT constructs IL1β-S2, IL1β-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd(3+) as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.

We report the direct dynamic nuclear polarization (DNP) of (13)C nuclei of a uniformly [(13)C,(15)N]-labeled, paramagnetic full-length hammerhead ribozyme (HHRz) complex with Mn(2+) where the enhanced polarization is fully provided by the endogenously bound metal ion and no exogenous polarizing agent is added. A (13)C enhancement factor of ε = 8 was observed by intra-complex DNP at 9.4 T. In contrast, "conventional" indirect and direct DNP experiments were performed using AMUPol as polarizing agent where we obtained a (1)H enhancement factor of ε ≈ 250. Comparison with the diamagnetic (Mg(2+)) HHRz complex shows that the presence of Mn(2+) only marginally influences the (DNP-enhanced) NMR properties of the RNA. Furthermore two-dimensional correlation spectra ((15)N-(13)C and (13)C-(13)C) reveal structural inhomogeneity in the frozen, amorphous state indicating the coexistence of several conformational states. These demonstrations of intra-complex DNP using an endogenous metal ion as well as DNP-enhanced MAS NMR of RNA in general yield important information for the development of new methods in structural biology.

Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3' end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

An algorithm, CYLIB, is presented for converting molecular topology descriptions from the PDB Chemical Component Dictionary into CYANA residue library entries. The CYANA structure calculation algorithm uses torsion angle molecular dynamics for the efficient computation of three-dimensional structures from NMR-derived restraints. For this, the molecules have to be represented in torsion angle space with rotations around covalent single bonds as the only degrees of freedom. The molecule must be given a tree structure of torsion angles connecting rigid units composed of one or several atoms with fixed relative positions. Setting up CYANA residue library entries therefore involves, besides straightforward format conversion, the non-trivial step of defining a suitable tree structure of torsion angles, and to re-order the atoms in a way that is compatible with this tree structure. This can be done manually for small numbers of ligands but the process is time-consuming and error-prone. An automated method is necessary in order to handle the large number of different potential ligand molecules to be studied in drug design projects. Here, we present an algorithm for this purpose, and show that CYANA structure calculations can be performed with almost all small molecule ligands and non-standard amino acid residues in the PDB Chemical Component Dictionary.

The automated assignment of NOESY cross peaks has become a fundamental technique for NMR protein structure analysis. A widely used algorithm for this purpose is implemented in the program CYANA. It has been used for a large number of structure determinations of proteins in solution but was so far not described in full detail. In this paper we present a complete description of the CYANA implementation of automated NOESY assignment, which differs extensively from its predecessor CANDID by the use of a consistent probabilistic treatment, and we discuss its performance in the second round of the critical assessment of structure determination by NMR.

Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in (15)N or (13)C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in (1)H-(15)N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.

The automated assignment of NOESY cross peaks has become a fundamental technique for NMR protein structure analysis. A widely used algorithm for this purpose is implemented in the program CYANA. It has been used for a large number of structure determinations of proteins in solution but a systematic evaluation of its performance has not yet been reported. In this paper we systematically analyze the reliability of combined automated NOESY assignment and structure calculation with CYANA under a variety of conditions on the basis of the experimental NMR data sets of ten proteins. To evaluate the robustness of the algorithm, the original high-quality experimental data sets were modified in different ways to simulate the effect of data imperfections, i.e. incomplete or erroneous chemical shift assignments, missing NOESY cross peaks, inaccurate peak positions, inaccurate peak intensities, lower dimensionality NOESY spectra, and higher tolerances for the matching of chemical shifts and peak positions. The results show that the algorithm is remarkably robust with regard to imperfections of the NOESY peak lists and the chemical shift tolerances but susceptible to lacking or erroneous resonance assignments, in particular for nuclei that are involved in many NOESY cross peaks.

The central hallmark of prion diseases is the misfolding of cellular prion protein (PrP(C)) into a disease-associated aggregated isoform known as scrapie prion protein (PrP(Sc)). NMR spectroscopy has made many essential contributions to the characterization of recombinant PrP in its folded, unfolded and aggregated states. Recent studies reporting on de novo generation of prions from recombinant PrP and infection of animals using prion aerosols suggest that adjustment of current biosafety measures may be necessary, particularly given the relatively high protein concentrations required for NMR applications that favor aggregation. We here present a protocol for the production of recombinant PrP under biosafety level 2 conditions that avoids entirely exposure of the experimenter to aerosols that might contain harmful PrP aggregates. In addition, we introduce an NMR sample tube setup that allows for safe handling of PrP samples at the spectrometer that usually is not part of a dedicated biosafety level 2 laboratory.

Protein structure determination by NMR can in principle be speeded up both by reducing the measurement time on the NMR spectrometer and by a more efficient analysis of the spectra. Here we study the reliability of protein structure determination based on a single type of spectra, namely nuclear Overhauser effect spectroscopy (NOESY), using a fully automated procedure for the sequence-specific resonance assignment with the recently introduced FLYA algorithm, followed by combined automated NOE distance restraint assignment and structure calculation with CYANA. This NOESY-FLYA method was applied to eight proteins with 63-160 residues for which resonance assignments and solution structures had previously been determined by the Northeast Structural Genomics Consortium (NESG), and unrefined and refined NOESY data sets have been made available for the Critical Assessment of Automated Structure Determination of Proteins by NMR project. Using only peak lists from three-dimensional (13)C- or (15)N-resolved NOESY spectra as input, the FLYA algorithm yielded for the eight proteins 91-98 % correct backbone and side-chain assignments if manually refined peak lists are used, and 64-96 % correct assignments based on raw peak lists. Subsequent structure calculations with CYANA then produced structures with root-mean-square deviation (RMSD) values to the manually determined reference structures of 0.8-2.0 Å if refined peak lists are used. With raw peak lists, calculations for 4 proteins converged resulting in RMSDs to the reference structure of 0.8-2.8 Å, whereas no convergence was obtained for the four other proteins (two of which did already not converge with the correct manual resonance assignments given as input). These results show that, given high-quality experimental NOESY peak lists, the chemical shift assignments can be uncovered, without any recourse to traditional through-bond type assignment experiments, to an extent that is sufficient for calculating accurate three-dimensional structures.

Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218-289) and α-synuclein yielded 88-97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77-90 % correctness if also assignments classified as tentative by the algorithm are included.

Peak overlap is one of the major factors complicating the analysis of biomolecular NMR spectra. We present a general method for predicting the extent of peak overlap in multidimensional NMR spectra and its validation using both, experimental data sets and Monte Carlo simulation. The method is based on knowledge of the magnetization transfer pathways of the NMR experiments and chemical shift statistics from the Biological Magnetic Resonance Data Bank. Assuming a normal distribution with characteristic mean value and standard deviation for the chemical shift of each observable atom, an analytic expression was derived for the expected overlap probability of the cross peaks. The analytical approach was verified to agree with the average peak overlap in a large number of individual peak lists simulated using the same chemical shift statistics. The method was applied to eight proteins, including an intrinsically disordered one, for which the prediction results could be compared with the actual overlap based on the experimentally measured chemical shifts. The extent of overlap predicted using only statistical chemical shift information was in good agreement with the overlap that was observed when the measured shifts were used in the virtual spectrum, except for the intrinsically disordered protein. Since the spectral complexity of a protein NMR spectrum is a crucial factor for protein structure determination, analytical overlap prediction can be used to identify potentially difficult proteins before conducting NMR experiments. Overlap predictions can be tailored to particular classes of proteins by preparing statistics from corresponding protein databases. The method is also suitable for optimizing recording parameters and labeling schemes for NMR experiments and improving the reliability of automated spectra analysis and protein structure determination.

Peak lists are commonly used in NMR as input data for various software tools such as automatic assignment and structure calculation programs. Inconsistencies of chemical shift referencing among different peak lists or between peak and chemical shift lists can cause severe problems during peak assignment. Here we present a simple and robust tool to achieve self-consistency of the chemical shift referencing among a set of peak lists. The Peakmatch algorithm matches a set of peak lists to a specified reference peak list, neither of which have to be assigned. The chemical shift referencing offset between two peak lists is determined by optimizing an assignment-free match score function using either a complete grid search or downhill simplex optimization. It is shown that peak lists from many different types of spectra can be matched reliably as long as they contain at least two corresponding dimensions. Using a simulated peak list, the Peakmatch algorithm can also be used to obtain the optimal agreement between a chemical shift list and experimental peak lists. Combining these features makes Peakmatch a useful tool that can be applied routinely before automatic assignment or structure calculation in order to obtain an optimized input data set.

A method is introduced to represent an ensemble of conformers of a protein by a single structure in torsion angle space that lies closest to the averaged Cartesian coordinates while maintaining perfect covalent geometry and on average equal steric quality and an equally good fit to the experimental (e.g. NMR) data as the individual conformers of the ensemble. The single representative 'regmean structure' is obtained by simulated annealing in torsion angle space with the program CYANA using as input data the experimental restraints, restraints for the atom positions relative to the average Cartesian coordinates, and restraints for the torsion angles relative to the corresponding principal cluster average values of the ensemble. The method was applied to 11 proteins for which NMR structure ensembles are available, and compared to alternative, commonly used simple approaches for selecting a single representative structure, e.g. the structure from the ensemble that best fulfills the experimental and steric restraints, or the structure from the ensemble that has the lowest RMSD value to the average Cartesian coordinates. In all cases our method found a structure in torsion angle space that is significantly closer to the mean coordinates than the alternatives while maintaining the same quality as individual conformers. The method is thus suitable to generate representative single structure representations of protein structure ensembles in torsion angle space. Since in the case of NMR structure calculations with CYANA the single structure is calculated in the same way as the individual conformers except that weak positional and torsion angle restraints are added, we propose to represent new NMR structures by a 'regmean bundle' consisting of the single representative structure as the first conformer and all but one original individual conformers (the original conformer with the highest target function value is discarded in order to keep the number of conformers in the bundle constant). In this way, analyses that require a single structure can be carried out in the most meaningful way using the first model, while at the same time the additional information contained in the ensemble remains available.

Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with (15)N-(1)H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three different species of amino acids in each sample, (15)N, 1-(13)C, and fully (13)C/(15)N labeled, permits identification of more amino acid types and sequential pairs than would be possible with previously published combinatorial methods. The new protocol involves recording of up to five 2D triple-resonance experiments to distinguish the various isotopomeric dipeptide species. The pattern of backbone NH cross peaks in this series of spectra adds a new dimension to the combinatorial grid, which otherwise mostly relies on comparison of [(15)N, (1)H]-HSQC and possibly 2D HN(CO) spectra of samples with different labeled amino acid compositions. Application to two α-helical membrane proteins shows that using no more than three samples information can be accumulated such that backbone assignments can be completed solely based on 3D HNCA/HN(CO)CA experiments. Alternatively, in the case of severe signal overlap in certain regions of the standard suite of triple-resonance spectra acquired on uniformly labeled protein, or missing signals due to a lack of efficiency of 3D experiments, the remaining gaps can be filled.

An extension to HN(CO-α/β-N,C(α)-J)-TROSY (Permi and Annila in J Biomol NMR 16:221-227, 2000) is proposed that permits the simultaneous determination of the four coupling constants (1) J (N'(i)Cα(i)), (2) J (HN(i)Cα(i)), (2) J (Cα(i-1)N'(i)), and (3) J (Cα(i-1)HN(i)) in (15)N,(13)C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the (2) J (CαN') coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both (2) J (CαN') and (1) J (N'Cα) (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.

A fully automated method is presented for determining NMR solution structures of proteins using exclusively NOESY spectra as input, obviating the need to measure any spectra only for obtaining resonance assignments but devoid of structural information. Applied to two small proteins, the approach yielded structures that coincided closely with conventionally determined structures.

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273-6279 (1982)), types of amino acids are labeled with (13)C or/and (15)N such that cross peaks between (13)CO(i - 1) and (15)NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with (13)C and the second with (15)N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B(2)R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.

We have analyzed the relaxation properties of all (31)P nuclei in an RNA cUUCGg tetraloop model hairpin at proton magnetic field strengths of 300, 600 and 900 MHz in solution. Significant H, P dipolar contributions to R (1) and R (2) relaxation are observed in a protonated RNA sample at 600 MHz. These contributions can be suppressed using a perdeuterated RNA sample. In order to interpret the (31)P relaxation data (R (1), R (2)), we measured the (31)P chemical shift anisotropy (CSA) by solid-state NMR spectroscopy under various salt and hydration conditions. A value of 178.5 ppm for the (31)P CSA in the static state (S (2) = 1) could be determined. In order to obtain information about fast time scale dynamics we performed a modelfree analysis on the basis of our relaxation data. The results show that subnanosecond dynamics detected around the phosphodiester backbone are more pronounced than the dynamics detected for the ribofuranosyl and nucleobase moieties of the individual nucleotides (Duchardt and Schwalbe, J Biomol NMR 32:295-308, 2005; Ferner et al., Nucleic Acids Res 36:1928-1940, 2008). Furthermore, the dynamics of the individual phosphate groups seem to be correlated to the 5' neighbouring nucleobases.

Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly 13C/15N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional "through-bond" spectrum (and 2D HSQC spectra) in addition to the 13C-edited and 15N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods.

A novel NMR pulse sequence has been developed that correlates the H2 resonances with the C2 and the N1 (N3) resonances in adenine nucleobases of 13C, 15N labeled oligonucleotides. The pulse scheme of the new 3D-HNHC experiment is composed of a 2J-15N-HSQC and a 1J-13C-HSQC and utilizes large 2J(H2, N1(N3)) and 1J(H2, C2) couplings. The experiment was applied to a medium-size 13C, 15N-labeled 36mer RNA. It is useful to resolve assignment ambiguities occurring especially in larger RNA molecules due to resonance overlap in the 1H-dimension. Therefore, the missing link in correlating the imino H3 resonances of the uracils across the AU base pair to the H8 resonances of the adenines via the novel pulse sequence and the TROSY relayed HCCH-COSY (Simon et al. in J Biomol NMR 20:173-176 2001) is provided.

High amino acid coverage labeling of the mammalian G protein coupled receptors (GPCR) rhodopsin was established with 15N and 15N/13C isotopes. Rhodopsin was expressed at preparative scale in HEK293S cells and studied in full-length by NMR spectroscopy in detergent micelle solution. This resulted in the assignment and detailed study of the dynamic properties of the C-terminus of rhodopsin. The rhodopsin C-terminus is immobilized until Ala333, after which it becomes unstructured.

A novel NMR pulse sequence is introduced to determine the glycosidic torsion angle chi in (13)C,(15)N-labeled oligonucleotides. The quantitative Gamma-HCNCH measures the dipolar cross-correlated relaxation rates Gamma(DD,DD)(C8H8,C1'H1') (pyrimidines) and Gamma(DD,DD)(C6H6,C1'H1') (purines). Cross-correlated relaxation rates of a (13)C,(15)N-labeled RNA 14mer containing a cUUCGg tetraloop were determined and yielded chi-angles that agreed remarkably well with data derived from the X-ray structure of the tetraloop. In addition, the method was applied to the larger stemloop D (SLD) subdomain of the Coxsackievirus B3 cloverleaf 30mer RNA and the effect of anisotropic rotational motion was examined for this molecule. It could be shown that the chi-angle determination especially for nucleotides in the anti conformation was very accurate and the method was ideally suited to distinguish between the syn and the anti-conformation of all four types of nucleotides.

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of alpha,epsilon-15N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state 13C,15N-REDOR and HETCOR experiments of all possible 13C'(i-1) carbonyl/15N(i)-tryptophan isotope labeled amide pairs, and H/D exchange 1H,15N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone 15N nuclei and partially to their bound protons. 1H,15N chemical shift assignment was achieved for indole side chains of Trp35(1.30) and Trp175(4.65). 15N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175(4.65) at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.

Megalin, an approx. 600 kDa transmembrane glycoprotein that acts as multi-ligand transporter, is a member of the low density lipoprotein receptor gene family. Several cysteine-rich repeats, each consisting of about 40 residues, are responsible for the multispecific binding of ligands. The solution structure of the twelfth cysteine-rich ligand-binding repeat with class A motif found in megalin features two short beta-strands and two helical turns, yielding the typical fold with a I-III, II-V and IV-VI disulfide bridge connectivity pattern and a calcium coordination site at the C-terminal end. The resulting differences in electrostatic surface potential compared to other ligand-binding modules of this gene family, however, may be responsible for the functional divergence.

Biophysical studies on membrane proteins by solid state NMR (SSNMR) can be carried out directly in a membrane environment. Samples are usually prepared in form of multi-lamellar dispersions for magic angle sample spinning or as aligned multi-layers for orientation dependent NMR experiments without sample rotation. A new development is the application of MAS NMR to aligned samples (MAOSS; Magic Angle Oriented Sample Spinning). In combination with separated local field (SLF) experiments, size and orientation of heteronuclear dipolar couplings may be extracted from two-dimensional experiments which correlate dipolar couplings with isotropic chemical shifts. The orientation of these (1)H-X dipolar couplings can be directly related to the orientation of molecular groups in the sample. Here, we demonstrate the feasibility of these experiments on highly ordered polyethylene fibers which serve as model compound. Based on these data, the experiment is also applied to ordered multi-layers of bacteriorhodopsin (purple membrane) which is used as a model for aligned membrane proteins. We present a detailed analysis of different experimental designs with respect to angular sensitivity and the influence of residual sample disorder ("mosaic spread"). The results of the MAOSS-SLF experiment are discussed within the context of established solid state NMR experiments which are usually performed without sample rotation and we compare the data to orientation information obtained from X-ray diffraction.

The characterization of unfolded states of proteins has recently attracted considerable interest, as the residual structure present in these states may play a crucial role in determining their folding and misfolding behavior. Here, we investigated the dynamics in the denatured state of ubiquitin in 8 M urea at pH2. Under these conditions, ubiquitin does not have any detectable local residual structure, and uniform 15N relaxation rates along the sequence indicate the absence of motional restrictions caused by residual secondary structure and/or long-range interactions. A comparison of different models to predict relaxation data in unfolded proteins suggests that the subnanosecond dynamics in unfolded states depend on segmental motions only and do not show a dependence on the residue type but for proline and glycine residues.

A major time-consuming step of protein NMR structure determination is the generation of reliable NOESY cross peak lists which usually requires a significant amount of manual interaction. Here we present a new algorithm for automated peak picking involving wavelet de-noised NOESY spectra in a process where the identification of peaks is coupled to automated structure determination. The core of this method is the generation of incremental peak lists by applying different wavelet de-noising procedures which yield peak lists of a different noise content. In combination with additional filters which probe the consistency of the peak lists, good convergence of the NOESY-based automated structure determination could be achieved. These algorithms were implemented in the context of the ARIA software for automated NOE assignment and structure determination and were validated for a polysulfide-sulfur transferase protein of known structure. The procedures presented here should be commonly applicable for efficient protein NMR structure determination and automated NMR peak picking.

A new protocol is described for the isotope (15N and 13C,15N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. 1H,13C and 15N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the Cgamma resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.

The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with 13C and 15N were studied by 13C spin relaxation experiments. R1, R1rho and the 13C-[1H] steady-state NOE of C6 and C1' in pyrimidine and C8 and C1' in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time tau(c) of 2.31 +/- 0.13 ns and an axial ratio of 1.35 +/- 0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported 13C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539-10551; Fiala et al., 2000 J. Biomol. NMR 16, 291-302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165-190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S2 < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S2 = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of 13C relaxation rates were found to be in agreement with 15N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702-709).

Triple-resonance two-dimensional H6/H5(C4N)H and C6/C5(C4N)H experiments are described that provide through-bond H6/H5 or C6/C5 to imino/amino correlations in pyrimidine bases in (13)C,(15)N-labeled RNA. The experiments simultaneously transfer H6/H5 magnetization by an INEPT step to the C6/C5 nuclei and by homonuclear CC- and heteronuclear CN-TOCSY steps via the intervening C4 nucleus to the N3/N4 nuclei and then by a reverse INEPT step to the imino/amino hydrogens. The sensitivity of these experiments is high as demonstrated using a 30-nucleotide pyrimidine rich RNA at a concentration of 0.9 mM at temperatures of 10 degrees C and 25 degrees C. This indicates the general applicability of the experiments and the possibility to obtain correlations for imino resonances in non-canonical regions of the target RNA.

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30-40 kDa to overcome problems arising from rapid (1)H and (13)C transverse relaxation. However, (1)H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D(2)O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H(2)O. As a side-effect partial C(alpha) protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary (1)H(N)- and (1)H(alpha)-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D(2)O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of C(alpha) protonation of individual amino acids is determined quantitatively and transverse relaxation properties of (1)H(N) and (15)N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.