Long-range 19F-15N distance measurements in highly-13C, 15N-enriched solid proteins with 19F-dephased REDOR shift (FRESH) spectroscopy

We present a novel rotational-echo double resonance (REDOR) method for detection of multiple (19)F-(15)N distances in solid proteins. The method is applicable to protein samples containing a single (19)F label, in addition to high levels of (13)C and (15)N enrichment. REDOR dephasing pulses are applied on the (19)F channel during an indirect constant time chemical shift evolution period on (15)N, and polarization is then transferred to (13)C for detection, with high-power (1)H decoupling throughout the sequence. This four-channel experiment reports site-specifically on (19)F-(15)N distances, with highly accurate determinations of approximately 5 A distances and detection of correlations arising from internuclear distances of at least 8 A. We demonstrate the method on the well-characterized 56-residue model protein GB1, where the sole tryptophan residue (Trp-43) has been labeled with 5-(19)F-Trp, in a bacterial growth medium also including (13)C-glucose and (15)N ammonium chloride. In GB1, 11 distances are determined, all agreeing within 20% of the X-ray structure distances. We envision the experiment will be utilized to measure quantitative long-range distances for protein structure determination.     

Structure calculation from unambiguous long-range amide and methyl 1H-1H distance restraints for a microcrystalline protein with MAS solid-state NMR spectroscopy

Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 ?. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ～1.1 ?.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments. V. NMR chemical shielding analysis of N-formyl-serinamide,a model for polar side-chain containing peptides

Knowledge of chemical shift-structure relationships could greatly facilitate the NMR chemical shift assignment and structure refinement processes that occur during peptide/protein structure determination via NMR spectroscopy. To determine whether such correlations exist for polar side chain containing amino acid residues the serine dipeptide model, For-L-Ser-NH(2), was studied. Using the GIAO-RHF/6-31+G(d) and GIAO-RHF/TZ2P levels of theory the NMR chemical shifts of all hydrogen ((1)H(N), (1)H(alpha), (1)H(beta1), (1)H(beta2)), carbon ((13)C(alpha), (13)C(beta), (13)C') and nitrogen ((15)N) atoms have been computed for all 44 stable conformers of For-L-Ser-NH(2). An attempt was made to establish correlation between chemical shift of each nucleus and the major conformational variables (omega(0), phi, psi, omega(1), chi,(1) and chi(2)). At both levels of theory a linear correlation can be observed between (1)H(alpha)/phi, (13)C(alpha)/phi, and (13)C(alpha)/psi. These results indicate that the backbone and side-chain structures of For-L-Ser-NH(2) have a strong influence on its chemical shifts.     

High resolution 1H-detected solid-state NMR spectroscopy of protein aliphatic resonances: access to tertiary structure information

Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely (13)C,(13)C distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-(13)C glycerol or [2]-(13)C glycerol to dilute the (13)C spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-γ nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the (13)C or (15)N detected version of the experiment. We report here a new labeling scheme, which enables (1)H-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken α-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic (1)H resonances. We show further that (13)C resolved 3D-(1)H,(1)H correlation experiments yield access to long-range proton-proton distances in the protein.     

Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.     

Automated protein structure determination from NMR spectra

Fully automated structure determination of proteins in solution (FLYA) yields, without human intervention, three-dimensional protein structures starting from a set of multidimensional NMR spectra. Integrating existing and new software, automated peak picking over all spectra is followed by peak list filtering, the generation of an ensemble of initial chemical shift assignments, the determination of consensus chemical shift assignments for all (1)H, (13)C, and (15)N nuclei, the assignment of NOESY cross-peaks, the generation of distance restraints, and the calculation of the three-dimensional structure by torsion angle dynamics. The resulting, preliminary structure serves as additional input to the second stage of the procedure, in which a new ensemble of chemical shift assignments and a refined structure are calculated. The three-dimensional structures of three 12-16 kDa proteins computed with the FLYA algorithm coincided closely with the conventionally determined structures. Deviations were below 0.95 A for the backbone atom positions, excluding the flexible chain termini. 96-97% of all backbone and side-chain chemical shifts in the structured regions were assigned to the correct residues. The purely computational FLYA method is suitable for substituting all manual spectra analysis and thus overcomes a main efficiency limitation of the NMR method for protein structure determination.     

Hydrogen exchange study on the hydroxyl groups of serine and threonine residues in proteins and structure refinement using NOE restraints with polar side-chain groups

We recently developed new NMR methods for monitoring the hydrogen exchange rates of tyrosine hydroxyl (Tyr-OH) and cysteine sulfhydryl (Cys-SH) groups in proteins. These methods facilitate the identification of slowly exchanging polar side-chain protons in proteins, which serve as sources of NOE restraints for protein structure refinement. Here, we have extended the methods for monitoring the hydrogen exchange rates of the OH groups of serine (Ser) and threonine (Thr) residues in an 18.2 kDa protein, EPPIb, and thus demonstrated the usefulness of NOE restraints with slowly exchanging OH protons for refining the protein structure. The slowly exchanging Ser/Thr-OH groups were readily identified by monitoring the (13)C(β)-NMR signals in an H(2)O/D(2)O (1:1) mixture, for the protein containing Ser/Thr residues with (13)C, (2)H-double labels at their β carbons. Under these circumstances, the OH groups exist in equilibrium between the protonated and deuterated isotopomers, and the (13)C(β) peaks of the two species are resolved when their exchange rate is slower than the time scale of the isotope shift effect. In the case of EPPIb dissolved in 50 mM sodium phosphate buffer (pH 7.5) at 40 °C, one Ser and four Thr residues were found to have slowly exchanging hydroxyl groups (k(ex) < ~40 s(-1)). With the information for the slowly exchanging Ser/Thr-OH groups in hand, we could collect additional NOE restraints for EPPIb, thereby making a unique and important contribution toward defining the spatial positions of the OH protons, and thus the hydrogen-bonding acceptor atoms.     

Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy

A major challenge for the structure determination of integral membrane proteins by solution NMR spectroscopy is the limited number of NOE restraints in these systems stemming from extensive deuteration. Paramagnetic relaxation enhancement (PRE) by means of nitroxide spin-labels can provide valuable long-range distance information but, in practice, has limits in its application to membrane proteins because spin-labels are often incompletely reduced in highly apolar environments. Using the integral membrane protein OmpA as a model system, we introduce a method of parallel spin-labeling with paramagnetic and diamagnetic labels and show that distances in the range 15-24 Angstroms can be readily determined. The protein was labeled at 11 water-exposed and lipid-covered sites, and 320 PRE distance restraints were measured. The addition of these restraints resulted in significant improvement of the calculated backbone structure of OmpA. Structures of reasonable quality can even be calculated with PRE distance restraints only, i.e., in the absence of NOE distance restraints.     

3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints

In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 x 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 A.     

Structure determination of protein-protein complexes using NMR chemical shifts: case of an endonuclease colicin-immunity protein complex

Nuclear magnetic resonance (NMR) spectroscopy provides a range of powerful techniques for determining the structures and the dynamics of proteins. The high-resolution determination of the structures of protein-protein complexes, however, is still a challenging problem for this approach, since it can normally provide only a limited amount of structural information at protein-protein interfaces. We present here the determination using NMR chemical shifts of the structure (PDB code 2K5X) of the cytotoxic endonuclease domain from bacterial toxin colicin (E9) in complex with its cognate immunity protein (Im9). In order to achieve this result, we introduce the CamDock method, which combines a flexible docking procedure with a refinement that exploits the structural information provided by chemical shifts. The results that we report thus indicate that chemical shifts can be used as structural restraints for the determination of the conformations of protein complexes that are difficult to obtain by more standard NMR approaches.     

Constant-time through-bond 13C correlation spectroscopy for assigning protein resonances with solid-state NMR spectroscopy

Even as available magnetic fields for NMR continue to increase, resolution remains one of the most critical limitations in assigning and solving structures of larger biomolecules. Here we present a novel constant-time through-bond correlation spectroscopy for solids that offers superior resolution for 13C chemical shift assignments in proteins. In this experiment, the indirect evolution and transfer periods are combined into a single constant time interval, offering increased resolution while not sacrificing sensitivity. In GB1, this allows us to resolve peaks that are otherwise unresolved and to make assignments in the absence of multibond transfers.     

Refinement of NMR structures using implicit solvent and advanced sampling techniques

NMR biomolecular structure calculations exploit simulated annealing methods for conformational sampling and require a relatively high level of redundancy in the experimental restraints to determine quality three-dimensional structures. Recent advances in generalized Born (GB) implicit solvent models should make it possible to combine information from both experimental measurements and accurate empirical force fields to improve the quality of NMR-derived structures. In this paper, we study the influence of implicit solvent on the refinement of protein NMR structures and identify an optimal protocol of utilizing these improved force fields. To do so, we carry out structure refinement experiments for model proteins with published NMR structures using full NMR restraints and subsets of them. We also investigate the application of advanced sampling techniques to NMR structure refinement. Similar to the observations of Xia et al. (J.Biomol. NMR 2002, 22, 317-331), we find that the impact of implicit solvent is rather small when there is a sufficient number of experimental restraints (such as in the final stage of NMR structure determination), whether implicit solvent is used throughout the calculation or only in the final refinement step. The application of advanced sampling techniques also seems to have minimal impact in this case. However, when the experimental data are limited, we demonstrate that refinement with implicit solvent can substantially improve the quality of the structures. In particular, when combined with an advanced sampling technique, the replica exchange (REX) method, near-native structures can be rapidly moved toward the native basin. The REX method provides both enhanced sampling and automatic selection of the most native-like (lowest energy) structures. An optimal protocol based on our studies first generates an ensemble of initial structures that maximally satisfy the available experimental data with conventional NMR software using a simplified force field and then refines these structures with implicit solvent using the REX method. We systematically examine the reliability and efficacy of this protocol using four proteins of various sizes ranging from the 56-residue B1 domain of Streptococcal protein G to the 370-residue Maltose-binding protein. Significant improvement in the structures was observed in all cases when refinement was based on low-redundancy restraint data. The proposed protocol is anticipated to be particularly useful in early stages of NMR structure determination where a reliable estimate of the native fold from limited data can significantly expedite the overall process. This refinement procedure is also expected to be useful when redundant experimental data are not readily available, such as for large multidomain biomolecules and in solid-state NMR structure determination.     

2D NMR方法研究抗癌药物冬凌草乙素的结构与谱线归属

用异核化学位移相关谱、远程异核化学位移相关谱和同核化学位移相关谱等现代核磁共振技术对抗癌中草药冬凌草中分离出的抗癌、抗菌有效成分冬凌草乙素分子的~(13)C和~1H化学位移进行了完全归属,为冬凌草乙素分子溶液中的三维空间结构研究提供了可靠的结构参数。     

On Restraints in End-Point Protein–Ligand Binding Free Energy Calculations

The impact of harmonic restraints on protein heavy atoms and ligand atoms on end-point free energy calculations is systematically characterized for 54 protein–ligand complexes. We observe that stronger restraints reduce the equilibration time and statistical inefficiency, suppress conformational sampling, influence correlation with experiment, and monotonically decrease the estimated loss of entropy upon binding, leading to stronger estimated binding free energies in most systems. A statistical estimator that reweights for the biasing potential and includes data prior to the estimated equilibration time has the highest correlation with experiment. A spring constant of 20 cal mol⁻¹ Å⁻² maintains a near-native energy landscape and suppresses artifactual energy minima while minimally limiting thermal fluctuations about the crystal structure. © 2019 Wiley Periodicals, Inc.     

NMR study of the spatial structure of synthetic pyrethroids

The spatial isomers of the new synthetic analogs of ethyl permithrinic ether and permethrin were investigated by NMR (¹H, ¹³C, DEPT (distortionless enhancement by polarization transfer), COSY (correlation spectroscopy), CHCORR (heteronuclear (C, H) shift correlation spectroscopy), ROESY (rotating-frame Overhauser effect spectroscopy)). Several tendencies were revealed in the ¹H and ¹³C chemical shifts of the α atoms of the substituents in the 2nd and 3rd positions of the cyclopropane ring. For substituents cis-orientated relative to the ester group, the spectra show a paramagnetic shift of the ¹H signals and the diamagnetic shift of the ¹³C signals relative to the trans-orientated substituents. The ¹H and ¹³C chemical shifts of the α atoms of the substituents in the 2nd and 3rd positions of the cyclopropane ring permit an unambiguous determination of the stereochemistry of ethyl permethrinic ether and permethrin analogs.     

G-matrix Fourier transform NOESY-based protocol for high-quality protein structure determination

A protocol for high-quality structure determination based on G-matrix Fourier transform (GFT) NMR is presented. Five through-bond chemical shift correlation experiments providing 4D and 5D spectral information at high digital resolution are performed for resonance assignment. These are combined with a newly implemented (4,3)D GFT NOESY experiment which encodes information of 4D 15N/15N-, 13C(alipahtic)/15N-, and 13C(aliphatic)/13C(aliphatic)-resolved [1H,1H]-NOESY in two subspectra, each containing one component of chemical shift doublets arising from 4D --> 3D projection at omega1:Omega(1H) +/- Omega(X) [X = 15N,13C(aliphatic)]. The peaks located at the centers of the doublets are obtained from simultaneous 3D 15N/13C(aliphatic)/13C(aromatic)-resolved [1H,1H]-NOESY, wherein NOEs detected on aromatic protons are also obtained. The protocol was applied for determining a high-quality structure of the 14 kDa Northeast Structural Genomics consortium target protein, YqfB (PDB ID ). Through-bond correlation and NOESY spectra were acquired, respectively, in 16.9 and 39 h (30 h for shift doublets, 9 h for central peaks) on a 600 MHz spectrometer equipped with a cryogenic probe. The rapidly collected highly resolved 4D NOESY information allows one to assign the majority of NOEs directly from chemical shifts, which yields accurate initial structures "within" approximately 2 angstroms of the final structure. Information theoretical "QUEEN" analysis of initial distance limit constraint networks revealed that, in contrast to structure-based protocols, such NOE assignment is not biased toward identifying additional constraints that tend to be redundant with respect to the available constraint network. The protocol enables rapid NMR data collection for robust high-quality structure determination of proteins up to approximately 20-25 kDa in high-throughput.     

On the use of pseudocontact shifts in the structure determination of metalloproteins

The utility of pseudocontact shifts in the structure refinement of metalloproteins has been evaluated using a native, paramagnetic Cu(2+) metalloprotein, plastocyanin from Anabaena variabilis (A.v.), as a model protein. First, the possibility of detecting signals of nuclei spatially close to the paramagnetic metal ion is investigated using the WEFT pulse sequence in combination with the conventional TOCSY and (1)H-(15)N HSQC sequences. Second, the importance of the electrical charge of the metal ion for the determination of correct pseudocontact shifts from the obtained chemical shifts is evaluated. Thus, using both the Cu(+) plastocyanin and Cd(2+)-substituted plastocyanin as the diamagnetic references, it is found that the Cd(2+)-substituted protein with the same electrical charge of the metal ion as the paramagnetic Cu(2+) plastocyanin provides the most appropriate diamagnetic reference signals. Third, it is found that reliable pseudocontact shifts cannot be obtained from the chemical shifts of the (15)N nuclei in plastocyanin, most likely because these shifts are highly dependent on even minor differences in the structure of the paramagnetic and diamagnetic proteins. Finally, the quality of the obtained (1)H pseudocontact shifts, as well as the possibility of improving the accuracy of the obtained structure, is demonstrated by incorporating the shifts as restraints in a refinement of the solution structure of A.v. plastocyanin. It is found that incorporation of the pseudocontact shifts enhances the precision of the structure in regions with only few NOE restraints and improves the accuracy of the overall structure.     

Complementary segmental labeling of large RNAs: economic preparation and simplified NMR spectra for measurement of more RDCs

NMR structure determination of large RNAs is often restricted by limited RDC information caused by chemical shift degeneracy. We established a general, time- and cost-effective methodology for the preparation of 13C/15N complementary labeled RNAs from a single plasmid. Applying this method to the 25 kDa BC1-DTE RNA, we were able to resolve severe chemical shift degeneracy, thereby almost doubling the number of RDC restraints in comparison to the conventional 13C,15N uniform-labeled RNA.     

Methyl proton contacts obtained using heteronuclear through-bond transfers in solid-state NMR spectroscopy

A two-dimensional proton-mediated carbon-carbon correlation experiment that relies on through-bond heteronuclear magnetization transfers is demonstrated in the context of solid-state NMR of proteins. This new experiment, dubbed J-CHHC by analogy to the previously developed dipolar CHHC techniques, is shown to provide selective and sensitive correlations in the methyl region of 2D spectra of crystalline organic compounds. The method is then demonstrated on a microcrystalline sample of the dimeric protein Crh (2 x 10.4 kDa). A total of 34 new proton-proton contacts involving side-chain methyl groups were observed in the J-CHHC spectrum, which had not been observed with the conventional experiment. The contacts were then used as additional distance restraints for the 3D structure determination of this microcrystalline protein. Upon addition of these new distance restraints, which are in large part located in the hydrophobic core of the protein, the root-mean-square deviation with respect to the X-ray structure of the backbone atom coordinates of the 10 best conformers of the new ensemble of structures is reduced from 1.8 to 1.1 A.     

Supramolecular interactions probed by 13C-13C solid-state NMR spectroscopy

We present a robust solid-state NMR approach for the accurate determination of molecular interfaces in insoluble and noncrystalline protein-protein complexes. The method relies on the measurement of intermolecular (13)C-(13)C distances in mixtures of [1-(13)C]glucose- and [2-(13)C]glucose-labeled proteins. We have applied this method to Parkinson's disease-associated α-synuclein fibrils and found that they are stacked in a parallel in-register arrangement. Additionally, intermolecular distance restraints for the structure determination of the fibrils at atomic resolution were measured.