Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination

X-ray crystallography and NMR spectroscopy provide the only sources of experimental data from which protein structures can be analyzed at high or even atomic resolution. The degree to which these methods complement each other as sources of structural knowledge is a matter of debate; it is often proposed that small proteins yielding high quality, readily analyzed NMR spectra are a subset of those that readily yield strongly diffracting crystals. We have examined the correlation between NMR spectral quality and success in structure determination by X-ray crystallography for 159 prokaryotic and eukaryotic proteins, prescreened to avoid proteins providing polydisperse and/or aggregated samples. This study demonstrates that, across this protein sample set, the quality of a protein's [15N-1H]-heteronuclear correlation (HSQC) spectrum recorded under conditions generally suitable for 3D structure determination by NMR, a key predictor of the ability to determine a structure by NMR, is not correlated with successful crystallization and structure determination by X-ray crystallography. These results, together with similar results of an independent study presented in the accompanying paper (Yee, et al., J. Am. Chem. Soc., accompanying paper), demonstrate that X-ray crystallography and NMR often provide complementary sources of structural data and that both methods are required in order to optimize success for as many targets as possible in large-scale structural proteomics efforts.     

Recent advances in protein NMR spectroscopy and their implications in protein therapeutics research

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the two main methods for protein three-dimensional structure determination at atomic resolution. According to the protein structures deposited in the Protein Data Bank, X-ray crystallography has become the dominant method for structure determination, particularly for large proteins and complexes. However, with the developments of isotope labeling, increase of magnetic field strength, common use of a cryogenic probe, and ingenious pulse sequence design, the applications of NMR spectroscopy have expanded in biological research, especially in characterizing protein dynamics, sparsely populated transient structures, weak protein interactions, and proteins in living cells at atomic resolution, which is difficult if not impossible by other biophysical methods. Although great advances have been made in protein NMR spectroscopy, its applications in protein therapeutics, which represents the fastest growing segment of the pharmaceutical industry, are still limited. Here we review the recent advances in the use of NMR spectroscopy in studies of large proteins or complexes, posttranslation modifications, weak interactions, and aggregation, and in-cell NMR spectroscopy. The potential applications of NMR spectroscopy in protein therapeutic assays are discussed.     

Crystallography Aided by Atomic Core-Level Binding Energies: Proton Transfer versus Hydrogen Bonding in Organic Crystal Structures

Ionic bond or hydrogen bridge? Br?nsted proton transfer to nitrogen acceptors in organic crystals causes strong N1s core-level binding energy shifts. A study of 15 organic cocrystal and salt systems shows that standard X-ray photoelectron spectroscopy (XPS) can be used as a complementary method to X-ray crystallography for distinguishing proton transfer from H-bonding in organic condensed matter.     

Application and limitations of X-ray crystallographic data in structure-based ligand and drug design

Structure-based design usually focuses upon the optimization of ligand affinity. However, successful drug design also requires the optimization of many other properties. The primary source of structural information for protein-ligand complexes is X-ray crystallography. The uncertainties introduced during the derivation of an atomic model from the experimentally observed electron density data are not always appreciated. Uncertainties in the atomic model can have significant consequences when this model is subsequently used as the basis of manual design, docking, scoring, and virtual screening efforts. Docking and scoring algorithms are currently imperfect. A good correlation between observed and calculated binding affinities is usually only observed only when very large ranges of affinity are considered. Errors in the correlation often exceed the range of affinities commonly encountered during lead optimization. Some structure-based design approaches now involve screening libraries by using technologies based on NMR spectroscopy and X-ray crystallography to discover small polar templates, which are used for further optimization. Such compounds are defined as leadlike and are also sought by more traditional high-throughput screening technologies. Structure-based design and HTS technologies show important complementarity and a degree of convergence.     

SMARTER crystallography of the fluorinated inorganic-organic compound Zn3Al2F12·[HAmTAZ]6

We present in this paper the structure resolution of a fluorinated inorganic-organic compound--Zn(3)Al(2)F(12)·[HAmTAZ](6)--by SMARTER crystallography, i.e. by combining powder X-ray diffraction crystallography, NMR crystallography and chemical modelling of crystal (structure optimization and NMR parameter calculations). Such an approach is of particular interest for this class of fluorinated inorganic-organic compound materials since all the atoms have NMR accessible isotopes ((1)H, (13)C, (15)N, (19)F, (27)Al, (67)Zn). In Zn(3)Al(2)F(12)·[HAmTAZ](6), (27)Al and high-field (19)F and (67)Zn NMR give access to the inorganic framework while (1)H, (13)C and (15)N NMR yield insights into the organic linkers. From these NMR experiments, parts of the integrant unit are determined and used as input data for the search of a structural model from the powder diffraction data. The optimization of the atomic positions and the calculations of NMR parameters ((27)Al and (67)Zn quadrupolar parameters and (19)F, (1)H, (13)C and (15)N isotropic chemical shifts) are then performed using a density functional theory (DFT) based code. The good agreement between experimental and DFT-calculated NMR parameters validates the proposed optimized structure. The example of Zn(3)Al(2)F(12)·[HAmTAZ](6) shows that structural models can be obtained in fluorinated hybrids by SMARTER crystallography on a polycrystalline powder with an accuracy similar to those obtained from single-crystal X-ray diffraction data.     

Absolute Configuration of Small Molecules by Co-Crystallization

The most reliable method to determine the absolute configuration of chiral molecules is X-ray crystallography, but small molecules can be difficult to crystallize. We report rapid co-crystallization of tetraaryladamantanes with small molecules as different as n-decane to nicotine to produce crystals for X-ray analysis and the assignment of absolute configuration when the molecules are chiral. A screen of 52 diverse compounds gave inclusion in co-crystals for 88 % of all cases and a high-resolution structure in 77 % of cases. Furthermore, starting from three milligrams of analyte, a combination of NMR spectroscopy and X-ray crystallography produced a full structure in less than three days using an adamantane crystallization chaperone that encapsulates the analyte at room temperature.     

Orientation of the axial ligands and magnetic properties of the hemes in the cytochrome c7 family from Geobacter sulfurreducens determined by paramagnetic NMR

Geobacter sulfurreducens is a sediment bacterium that contains a large number of multiheme cytochromes. The family of five c(7) triheme periplasmic cytochromes from Geobacter sulfurreducens shows structural diversity of the heme core. Structural characterization of the relative orientation of the axial ligands of these proteins by (13)C-paramagnetic NMR was carried out. The structures in solution were compared with those obtained by X-ray crystallography. For some hemes significant differences exist between the two methods such that orientation of the magnetic axes obtained from NMR data and the orientation taken from the X-ray coordinates differ. The results allowed the orientation of the magnetic axes to be defined confidently with respect to the heme frame in solution, a necessary step for the use of paramagnetic constraints to improve the complete solution structure of these proteins.     

X射线晶体学结合电子晶体学在复杂无机晶体结构解析中的应用

自然界中的材料,比如无机材料,有机材料,生物材料等等,均有其独特的物理和化学性质。而材料的性能又与材料的结构息息相关,只有充分了解了材料的结构,才能更加深入的研究材料性质。因此,材料结构的确定在化学、物理、生物等学科中的显得尤为重要。X射线晶体学作为传统的结构解析技术仍然是目前最重要的结构解析手段,但是对于复杂结构,X射线衍射晶体学解析结构也存在一些不足,往往需要其他技术手段相补充才能完成复杂结构的结构解析。电子晶体学虽然起步比X射线晶体学晚,但是,经过近几十年的发展,已经是结构解析领域一个非常重要的手段。本文将主要介绍X射线晶体学结合电子晶体学在复杂无机晶体结构解析中的应用。     

NMR and X-ray crystallography, complementary tools in structural proteomics of small proteins

NMR spectroscopy and X-ray crystallography, the two primary experimental methods for protein structure determination at high resolution, have different advantages and disadvantages in terms of sample preparation and data collection and analysis. It is therefore of interest to assess their complementarity when applied to small proteins. Structural genomics/proteomics projects provide an ideal opportunity to make such comparisons as they generate data in a systematic manner for large enough numbers of proteins to allow firm conclusions to be drawn. Here we report a comparison for 263 unique proteins screened by both NMR spectroscopy and X-ray crystallography in our structural proteomics pipeline. Only 21 targets (8%) were deemed amenable to both methods based on an initial 2D 15N-HSQC NMR spectrum and optimized crystallization trials. However, the use of both methods in the pipeline increased the total number of targets amenable to structure determination to 107, with 43 amenable to NMR only and 43 amenable to X-ray crystallographic methods only. We did not observe a correlation between 15N-HSQC spectral quality and the success of the same protein in crystallization screens. Similar results were found for an independent set of 159 proteins as reported in the accompanying paper by Snyder et al. Thus, we conclude that both methods are highly complementary, and in order to increase the number of proteins suited for structure determination, we suggest that both methods be used in parallel in screening of all small proteins for structure determination.     

Synthesis and properties of metal-ligand complexes with endohedral amine functionality

A series of tetracationic M(2)L(4) palladium-pyridyl complexes with endohedral amine functionality have been synthesized. The complexes were analyzed by NMR techniques (including Diffusion NMR and 2D NOESY), electrospray ionization (ESI) mass spectrometry, and X-ray crystallography. The solid state analysis shows a large change in crystal morphology upon introduction of the endohedral amine groups, caused by deleterious interactions between the amines and the triflate counterions from the coordination process. Combination of different ligands allows analysis of ligand exchange rates via NMR analysis, with half-lives on the order of 3 h, independent of the donor properties of the ligand. Self-sorting behavior is observed, with more electron-rich ligands being favored. The amine-containing and extended complexes are strongly fluorescent, giving quantum yields of up to 83%.     

Acetylacetonate bis(thiosemicarbazone) complexes of copper and nickel: towards new copper radiopharmaceuticals

A series of copper(II) and nickel(II) 1,3-bis(thiosemicarbazonato) complexes have been synthesised by the reaction of the metal acetates with pyrazoline proligands. In each case the complexes have an overall neutral charge with a dianionic ligand. The copper 1,3-bis(4-methyl-3-thiosemicarbazonato complex has been characterised by X-ray crystallography, which shows the copper is in an essentially square-planar symmetric N(2)S(2) environment. The nickel 1,3-bis(4-methyl-3-thiosemicarbazonato) and nickel 1,3-bis(4-phenyl-3-thiosemicarbazonato) complexes have been characterised by X-ray crystallography and show that in these cases the nickel is in a distorted square-planar environment, but the bonding mode of the ligands is unusual; the nickel binds to one of the aza-methinic nitrogen atoms and one hydrazinic nitrogen, creating one five-membered N-N-C-S-Ni chelate ring and one four-membered N-C-S-Ni chelate ring. Interestingly, the X-ray structure of the ethyl analogue [1,3-bis(4-ethyl-3-thiosemicarbazonato)nickel(II)] shows that in this case the nickel is symmetrically coordinated in the usual manner. The nickel complexes are diamagnetic and the different coordination modes are confirmed in solution by NMR spectroscopy. The complexes are susceptible to oxidation in air and a nickel complex, in which the central methylene carbon has been oxidised, has been characterised by X-ray crystallography and NMR spectroscopy. Electrochemical measurements show that the copper complexes undergo a reversible one-electron reduction at biologically accessible potentials.     

Paramagnetic NMR spectroscopy and density functional calculations in the analysis of the geometric and electronic structures of iron-sulfur proteins

Paramagnetic NMR spectroscopy has been underutilized in the study of metalloproteins. One difficulty of the technique is that paramagnetic relaxation broadens signals from nuclei near paramagnetic centers. In systems with low electronic relaxation rates, this makes such signals difficult to observe or impossible to assign by traditional methods. We show how the challenges of detecting and assigning signals from nuclei near the metal center can be overcome through the combination of uniform and selective 2H, 13C, and 15N isotopic labeling with NMR experiments that utilize direct one-dimensional (2H, 13C, and 15N) and two-dimensional (13C-X) detection. We have developed methods for calculating NMR chemical shifts and relaxation rates by density functional theory (DFT) approaches. We use the correspondence between experimental NMR parameters and those calculated from structural models of iron-sulfur clusters derived from X-ray crystallography to validate the computational approach and to investigate how structural differences are manifested in these values. We have applied this strategy to three iron-sulfur proteins: Clostridium pasteurianum rubredoxin, Anabaena [2Fe-2S] ferredoxin, and human [2Fe-2S] ferredoxin. Provided that an accurate structural model of the iron-sulfur cluster and surrounding residues is available from diffraction data, our results show that DFT calculations can return NMR observables with excellent accuracy. This suggests that it might be possible to use calculations to refine structures or to generate structural models of active sites when crystal structures are unavailable. The approach has yielded insights into the electronic structures of these iron-sulfur proteins. In rubredoxin, the results show that substantial unpaired electron spin is delocalized across NH...S hydrogen bonds and that the reduction potential can be changed by 77 mV simply by altering the strength of one of these hydrogen bonds. In reduced [2Fe-2S] ferredoxins, hyperfine shift data have provided quantitative information on the degree of valence trapping. The approach described here for iron-sulfur proteins offers new avenues for detailed studies of these and other metalloprotein systems.     

A Schiff-base porphyrin complex with double intramolecular hydrogen bonds

We have designed a porphyrin with a Schiff-base substituent as a model to study intramolecular hydrogen-bonding. The corresponding complex [Zn(SATPP)(CH₃OH)] has been synthesized and characterized by X-ray crystallography, ¹H NMR, and UV-Vis spectroscopy. The structure shows that there are three phenyl groups and one salicylideneaminophenyl group at the meso positions of the porphyrin, and the phenol oxygen is involved in double hydrogen bonds, one within the salicylideneaminophenyl and the other between coordinated methanol and phenol oxygen. ¹H NMR spectra suggest that the binding of methanol to zinc is an equilibrium process in solution and the equilibrium constant has been determined by UV-Vis measurements. The intramolecular hydrogen bond stabilizes the structure, and the binding affinity increases 10 times over the corresponding TPP (TPP, dianion of meso-5,10,15,20-tetraphenylporphyrin).     

The Respective Benefits of X-ray Crystallography and NMR for the Structural Determination of the Inclusion Complex Between Butyl-isothiocyanate and Alpha-cyclodextrin

Isothiocyanates are natural products extracted from plants. These molecules which exhibit very interesting antifungal properties, are insoluble in water. To increase their solubility, we have prepared inclusion complexes with different cyclodextrins. Among all the isothiocyanates studied, we have investigated in more detail the structure of one complex: butyl-isothiocyanate and alpha-cyclodextrin, using two different techniques. Firstly, ¹H NMR experiments were performed and revealed the inclusion phenomenon. In parallel, crystals of butyl-isothiocyanate–alpha-cyclodextrin were grown and their crystallographic structure determined. This confirmed the inclusion of the ITC molecules and allowed us to determine the exact position of the guest. Finally, we showed that even though the complex structure was determined separately in solution and in the solid state, the structural characterisations obtained with these two techniques are complementary, enhancing the respective benefits of X-ray crystallography and NMR.     

NMR Crystallography Enhanced by Quantum Chemical Calculations and Liquid State NMR Spectroscopy for the Investigation of Se-NHC Adducts**

N-heterocyclic carbene ligands (NHC) are widely utilized in catalysis and material science. They are characterized by their steric and electronic properties. Steric properties are usually quantified on the basis of their static structure, which can be determined by X-ray diffraction. The electronic properties are estimated in the liquid state; for example, via the ⁷⁷Se liquid state NMR of Se-NHC adducts. We demonstrate that ⁷⁷Se NMR crystallography can contribute to the characterization of the structural and electronic properties of NHC in solid and liquid states. Selected Se-NHC adducts are investigated via ⁷⁷Se solid state NMR and X-ray crystallography, supported by quantum chemical calculations. This investigation reveals a correlation between the molecular structure of adducts and NMR parameters, including not only isotropic chemical shifts but also the other chemical shift tensor components. Afterwards, the liquid state ⁷⁷Se NMR data is presented and interpreted in terms of the quantum chemistry modelling. The discrepancy between the structural and electronic properties, and in particular the π-accepting abilities of adducts in the solid and liquid states is discussed. Finally, the ¹³C isotropic chemical shift from the liquid state NMR and the ¹³C tensor components are also discussed, and compared with their ⁷⁷Se counterparts. ⁷⁷Se NMR crystallography can deliver valuable information about NHC ligands, and together with liquid state ⁷⁷Se NMR can provide an in-depth outlook on the properties of NHC ligands.     

Thermodynamic Differentiation of the Two Sides of Azabuckybowl through Complexation with Square Planar Platinum(II)

The precise control of the two faces, concave/convex faces, is an attractive challenge to realizing novel dynamic molecular systems. Herein, we report the synthesis, X-ray crystal structure, and bowl-to-bowl inversion behavior of a platinum complex with azabuckybowl as a monodentate ligand. X-ray crystallography revealed that the azabuckybowl is orthogonally coordinated to the plane containing the Pt center and other ligands. One and two-dimensional NMR studies have also confirmed that this complex was observed as mixtures of two isomers, although the isomeric ratio was highly biased. Theoretical calculations indicate that the difference in thermodynamic stability of these isomers is due to the direction of the concave/convex face of an azabuckybowl ligand.     

31P solid-state nuclear magnetic resonance study of the sorption of phosphate onto gibbsite and kaolinite

Sorption of phosphate onto gibbsite (gamma-Al(OH)3) and kaolinite has been studied by both macroscopic and 31P solid-state NMR measurements. Together these measurements indicate that phosphate is sorbed by a combination of surface complexation and surface precipitation with the relative amounts of these phases depending on pH and phosphate concentration. At low pH and high phosphate concentrations sorption is dominated by the presence of both amorphous and crystalline precipitate phases. The similarity between the single-pulse and CP/MAS NMR spectra suggests that the precipitate phases form a thin layer on the surface of the particles in close contact with protons from surface hydroxyl groups or coordinated water molecules. While the crystalline phase is only evident on samples below pH 7, amorphous AlPO4 was found at all pH and phosphate concentrations studied. As pH was increased the fraction of phosphate sorbed as an inner-sphere complex increased, becoming the dominant surface species by pH 8. Comparison of sorption and NMR results suggests that the inner-sphere complexes form by monodentate coordination to singly coordinated Al-OH sites on the edges of the gibbsite and kaolinite crystals. Outer-sphere phosphate complexes, which are readily desorbed, are also present at high pH.     

Rotational-echo double-resonance NMR distance measurements for the tubulin-bound Paclitaxel conformation

The important anticancer drug Taxol (paclitaxel, PTX) owes its unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. The conformation of the microtubule-bound drug has been the focus of numerous research efforts, since the inability of polymerized tubulin to form crystals precludes structure proof by X-ray crystallography. Likewise, although the alpha,beta-tubulin dimer structure has been solved by electron crystallography, the 3.7 A resolution is too low to permit direct determination of either ligand conformation or binding pose. In this article, we present experimental results from 2H{19F} REDOR NMR that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.     

Low Affinity Carbohydrate Lectin Interactions Examined with Surface Plasmon Resonance

The recognition of carbohydrates by proteins is an important element in many biological events like cell targeting, tumor invasion, immune response or bacterial and viral adhesion to host cells.^1,2 Besides structural data obtained from X-ray crystallography or NMR spectroscopy, the analysis of carbohydrate protein interactions should also take into consideration other biophysical parameters such as dissociation constants or kinetic rate constants of the complex. However, these parameters are often difficult to determine due to the rather low affinity of the systems under investigation.     

Dinuclear N-heterocyclic carbene complexes of silver(I), derived from imidazolium-linked cyclophanes

The synthesis of four new silver complexes of bidentate N-heterocyclic carbenes, derived from imidazolium-linked cyclophanes, has been achieved via a simple complexation reaction of the imidazolium-linked cyclophanes with the basic metal source Ag(2)O. The cyclophane structures contain two imidazolyl links between ortho-, meta- and mixed ortho/meta-substituted aromatic rings. The new silver carbene systems are thermally stable and have been characterised by NMR spectroscopy and X-ray crystallography. Three of the complexes have a dimeric structure of the form [L(2)Ag(2)](2+) in the solid state that is rigid on the NMR timescale in solution. The fourth complex has a neutral structure of the form [L(AgBr)(2)], the NMR studies suggesting some lability of the L-Ag bonding in solution.