Resolution-by-proxy: a simple measure for assessing and comparing the overall quality of NMR protein structures

In protein X-ray crystallography, resolution is often used as a good indicator of structural quality. Diffraction resolution of protein crystals correlates well with the number of X-ray observables that are used in structure generation and, therefore, with protein coordinate errors. In protein NMR, there is no parameter identical to X-ray resolution. Instead, resolution is often used as a synonym of NMR model quality. Resolution of NMR structures is often deduced from ensemble precision, torsion angle normality and number of distance restraints per residue. The lack of common techniques to assess the resolution of X-ray and NMR structures complicates the comparison of structures solved by these two methods. This problem is sometimes approached by calculating "equivalent resolution" from structure quality metrics. However, existing protocols do not offer a comprehensive assessment of protein structure as they calculate equivalent resolution from a relatively small number (<5) of protein parameters. Here, we report a development of a protocol that calculates equivalent resolution from 25 measurable protein features. This new method offers better performance (correlation coefficient of 0.92, mean absolute error of 0.28 ?) than existing predictors of equivalent resolution. Because the method uses coordinate data as a proxy for X-ray diffraction data, we call this measure "Resolution-by-Proxy" or ResProx. We demonstrate that ResProx can be used to identify under-restrained, poorly refined or inaccurate NMR structures, and can discover structural defects that the other equivalent resolution methods cannot detect. The ResProx web server is available at http://www.resprox.ca.     

Novel NMR tools to study structure and dynamics of biomembranes

Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13C-(1)H dipolar couplings, benefit from the excellent resolution of 13C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.     

X‐ray vs. NMR structures as templates for computational protein design

Certain protein‐design calculations involve using an experimentally determined high‐resolution structure as a template to identify new sequences that can adopt the same fold. This approach has led to the successful design of many novel, well‐folded, native‐like proteins. Although any atomic‐resolution structure can serve as a template in such calculations, most successful designs have used high‐resolution crystal structures. Because there are many proteins for which crystal structures are not available, it is of interest whether nuclear magnetic resonance (NMR) templates are also appropriate. We have analyzed differences between using X‐ray and NMR templates in side‐chain repacking and design calculations. We assembled a database of 29 proteins for which both a high‐resolution X‐ray structure and an ensemble of NMR structures are available. Using these pairs, we compared the rotamericity, χ₁‐angle recovery, and native‐sequence recovery of X‐ray and NMR templates. We carried out design using RosettaDesign on both types of templates, and compared the energies and packing qualities of the resulting structures. Overall, the X‐ray structures were better templates for use with Rosetta. However, for ～20% of proteins, a member of the reported NMR ensemble gave rise to designs with similar properties. Re‐evaluating RosettaDesign structures with other energy functions indicated much smaller differences between the two types of templates. Ultimately, experiments are required to confirm the utility of particular X‐ray and NMR templates. But our data suggest that the lack of a high‐resolution X‐ray structure should not preclude attempts at computational design if an NMR ensemble is available. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Solid-phase 31P NMR spectra of peat and mineral soils,humic acids and soil solution components: influence of iron and manganese

Solid-phase³¹P nuclear magnetic resonance (NMR) offers a direct means to determine the chemical environment of P present in soil and soil fractions. Iron is often a major component in soil and it has been thought that the presence of paramagnetic Fe and Mn in soil components is responsible for loss of resolution in NMR spectra. We have found that the resolution of signals in the solid-phase ³¹P NMR spectra of a Fe- and Mn-rich mineral soil was no worse than that for a series of four peat soils with a comparable concentration of P. Similarly, the resolution in the solid-phase ³¹P NMR spectra of the humic acid from the mineral soil was not much changed by extraction of the humic acid with acetylacetone in diethyl ether which removed around 40% of its Fe and 30% of its Mn. Removal of up to 50% of the Fe from organic rich, freeze-dried soil solutions from a soil catena with different land uses produced little change in spectral resolution. It was concluded that the limitations to resolution in solid-phase ³¹P NMR spectroscopy of soil humic substances do not stem from the presence of paramagnetic substances, but from the variable way P species are physically held in the amorphous milieu of the organic phase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detergent delipidation and solubilization strategies for high-resolution NMR of the membrane protein bacteriorhodopsin

High-resolution NMR studies of bacteriorhodopsin require the availability of the detergent-solubilized protein with both high concentration and small rotational correlation time. A procedure is described for the optimized preparation of such samples. Bacteriorhodopsin was first delipidated by detergent treatment of purple membrane under nonsolubilizing conditions for the protein. The delipidated aggregated protein could then be solubilized into monomers at concentration close to millimolar by selected detergents. The solubilizing detergent had an important effect on the rotational correlation time of the protein as shown by measuring in each case the temperature-dependent stability of the protein, the size of the detergent-protein complex, and the detergent viscosity. Consistently, a strong influence of the detergent was also found on spectral resolution in 13C NMR spectra of solubilized bacteriorhodopsin labeled with [1-13C]phenylalanine. Best resolution was obtained using n-dodecylmaltoside as detergent, with which relatively narrow well resolved 13C NMR resonances were observed at 50 degrees C. It is suggested that high-resolution NMR studies performed with this detergent may contribute to the structural resolution of bacteriorhodopsin.     

Use of fully deuterated micelles for conformational studies of membrane proteins by high resolution 1H nuclear magnetic resonance

Micellar complexes of melittin with fully deuterated detergents have been studied by high resolution ¹H nuclear magnetic resonance (NMR). The synthesis of deuterated micelles is described and it is shown that the ¹H NMR spectrum of micelle-bound melittin is well resolved and suitable for detailed analysis by conventional high-resolution NMR methods. A preliminary characterization of micelle-bound melittin shows that interaction with the micelle results in different conformational and dynamic features for the hydrophobic and hydrophilic regions of the melittin amino acid sequence. The present experiments on melittin and preliminary results with other polypeptides and proteins demonstrate that in favourable cases high-resolution ¹H NMR studies of the complexes formed between membrane proteins and deuterated micelles provides a viable method for conformational studies of membrane-bound proteins.     

Improving resolution in multidimensional NMR using random quadrature detection with compressed sensing reconstruction

NMR spectroscopy is central to atomic resolution studies in biology and chemistry. Key to this approach are multidimensional experiments. Obtaining such experiments with sufficient resolution, however, is a slow process, in part since each time increment in every indirect dimension needs to be recorded twice, in quadrature. We introduce a modified compressed sensing (CS) algorithm enabling reconstruction of data acquired with random acquisition of quadrature components in gradient-selection NMR. We name this approach random quadrature detection (RQD). Gradient-selection experiments are essential to the success of modern NMR and with RQD, a 50 % reduction in the number of data points per indirect dimension is possible, by only acquiring one quadrature component per time point. Using our algorithm (CS_RQD), high quality reconstructions are achieved. RQD is modular and combined with non-uniform sampling we show that this provides increased flexibility in designing sampling schedules leading to improved resolution with increasing benefits as dimensionality of experiments increases, with particular advantages for 4- and higher dimensional experiments.     

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.     

Protein resonance assignment by solid-state NMR based on 1H-detected 13C double-quantum spectroscopy at fast MAS

Journal of Biomolecular NMR - Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of...     

Comparison of NMR and crystal structures of membrane proteins and computational refinement to improve model quality

Membrane proteins are challenging to study and restraints for structure determination are typically sparse or of low resolution because the membrane environment that surrounds them leads to a variety of experimental challenges. When membrane protein structures are determined by different techniques in different environments, a natural question is “which structure is most biologically relevant?” Towards answering this question, we compiled a dataset of membrane proteins with known structures determined by both solution NMR and X‐ray crystallography. By investigating differences between the structures, we found that RMSDs between crystal and NMR structures are below 5 Å in the membrane region, NMR ensembles have a higher convergence in the membrane region, crystal structures typically have a straighter transmembrane region, have higher stereo‐chemical correctness, and are more tightly packed. After quantifying these differences, we used high‐resolution refinement of the NMR structures to mitigate them, which paves the way for identifying and improving the structural quality of membrane proteins.     

Application of linear prediction singular value decomposition for processingin vivo NMR data with low signal-to-noise ratio

Summary Low sensitivity of nuclear magnetic resonance (NMR) measurements of living cell composition by conventional methods requires samples with high cell density compared to that in growing cultures. Reasonably accurate intracellular concentration estimates from lower cell density samples can be obtained by treating the time-domain NMR data by linear prediction singular value decomposition (LPSVD) prior to Fourier transformation. Alternatively, application of LPSVD enables intracellular concentration estimates in less NMR acquisition time, improving time resolution in NMR measurements of intracellular transients.     

Extending the NMR spatial resolution limit for RNA by motional couplings

Experimental resolution of distinct dynamical processes in molecules can prove impossible when they are correlated to one another. In nuclear magnetic resonance (NMR) spectroscopy, couplings between internal and overall motions lead to intractable complexity, depriving insights into functionally important motions. Here we demonstrate that motional couplings can be used to anchor NMR frames of reference onto different parts of an RNA molecule, thus extending the spatial resolution limit for dynamical characterization.     

Phosphorus nuclear magnetic resonance of diverse phosvitin species

1. High resolution 31P nuclear magnetic resonance (NMR) spectra, with and without proton decoupling, of the principal egg phosphoproteins--phosvitins--of a bird (Gallus gallus), an amphibian (Xenopus laevis) and a fish (Salmo gairdneri) were obtained. 2. The spectra were evaluated with special reference to available amino acid sequences and the major NMR resonance in all three spectra was assigned to phosphoserine clusters. 3. The resolution of numerous additional phosphorus resonances provides the basis for further investigation of the particular molecular environments of phosvitin-bound phosphoryl groups and their involvement in the diverse binding modes for metal complex formation by phosvitins.     

Switched-angle spinning applied to bicelles containing phospholipid-associated peptides

In a model study, the proton NMR spectrum of the opioid pentapeptide leucine-enkephalin associated with bicelles is investigated. The spectral resolution for a static sample is limited due to the large number of anisotropic interactions, in particular strong proton–proton couplings, but resolution is greatly improved by magic-angle sample spinning. Here we present two-dimensional switched-angle spinning NMR experiments, which correlate the high-resolution spectrum of the membrane-bound peptide under magic-angle spinning with its anisotropic spectrum, leading to well-resolved spectra. The two-dimensional spectrum allows the exploitation of the high resolution of the isotropic spectrum, while retaining the structural information imparted by the anisotropic interactions in the static spectrum. Furthermore, switched-angle spinning techniques are demonstrated that allow one to record the proton spectrum of ordered bicellar phases as a function of the angle between the rotor axis and the magnetic field direction, thereby scaling the dipolar interactions by a predefined factor.     

A nuclear magnetic resonance imaging technique designed for studies of water in plant leaves.

A new imaging technique is described which uses nuclear magnetic resonance (NMR) to create a water profile of plant leaves. The water profile shows the average distribution of water as a function of depth into a leaf along a line perpendicular to the leaf surface; it can be used to measure the thickness of cell layers and the quantity of water in each layer. Two-dimensional NMR methods were used to avoid chemical shift distortion which degrades the resolution in leaf images made by conventional NMR techniques; image resolution was improved further by deconvolution analysis. To illustrate its application, the technique was used to follow changes in the internal structure of developing leaves.     

Hyperpolarized NMR metabolomics

Hyperpolarized NMR is a promising approach to address the sensitivity limits of conventional NMR metabolomics approaches, which currently fails to detect minute metabolite concentrations in biological samples. This review describes how tremendous signal enhancement offered by dissolution-dynamic nuclear polarization and parahydrogen-based techniques can be fully exploited for molecular omics sciences. Recent developments, including the combination of hyperpolarization techniques with fast multi-dimensional NMR implementation and quantitative workflows are described, and a comprehensive comparison of existing hyperpolarization techniques is proposed. High-throughput, sensitivity, resolution and other relevant challenges that should be tackled for a general application of hyperpolarized NMR in metabolomics are discussed.     

Solution NMR structure determination of proteins revisited

This 'Perspective' bears on the present state of protein structure determination by NMR in solution. The focus is on a comparison of the infrastructure available for NMR structure determination when compared to protein crystal structure determination by X-ray diffraction. The main conclusion emerges that the unique potential of NMR to generate high resolution data also on dynamics, interactions and conformational equilibria has contributed to a lack of standard procedures for structure determination which would be readily amenable to improved efficiency by automation. To spark renewed discussion on the topic of NMR structure determination of proteins, procedural steps with high potential for improvement are identified.     

Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.     

NMR imaging of roots: effects after root freezing of containerised conifer seedlings

Seedlings of Scots pine ( Pinus sylvestris L.) and Norway spruce [ Picea abies (L) Karst.] were subjected to low root temperatures, and 10 days later the roots were examined by NMR imaging. The amount of NMR detectable roots decreased with decreasing temperature, with the signal from the younger roots at the bottom of the container being the first to disappear. The origin of the loss of NMR signal is unclear but may be due to changes in the NMR properties of root water after cold damage. A recent method is discussed for obtaining unbiased estimates of root lengths from a series of total vertical projections; the method is particularly suited to evaluating NMR projection images. Since NMR imaging methods can apparently distinguish between control and cold damaged roots, it may be possible to design more routine applications using low resolution NMR methods.