The accuracy limit of chemical shift predictions for species in aqueous solution.

Interpreting NMR experiments benefits from first-principles predictions of chemical shifts. Reaching the accuracy limit of theory is relevant for unambiguous structural analysis and dissecting theoretical approximations. Since accurate chemical shift measurements are based on using internal reference compounds such as trimethylsilylpropanesulfonate (DSS), a detailed comparison of experimental with theoretical data requires simultaneous consideration of both target and reference species ensembles in the same solvent environment. Here we show that ab initio molecular dynamics simulations to generate liquid-state ensembles of target and reference compounds, including explicitly their short-range solvation environments and combined with quantum-mechanical solvation models, allows for predicting highly accurate 1H (∼0.1-0.5 ppm) and aliphatic 13C (∼1.5 ppm) chemical shifts for aqueous solutions of the model compounds trimethylamine N-oxide (TMAO) and N-methylacetamide (NMA), referenced to DSS without any system-specific adjustments. This encompasses the two peptide bond conformations of NMA identified by NMR. The results are used to derive a general-purpose guideline set for predictive NMR chemical shift calculations of NMA in the liquid state and to identify artifacts of force field models. Accurate predictions are only obtained if a sufficient number of explicit water molecules is included in the quantum-mechanical calculations, disproving a purely electrostatic model of the solvent effect on chemical shifts.

A lens-shaped supramolecule based on the bulky pentaphosphaferrocene [CpBIGFe(η5-P5)] and CuBr2.

Besides inherent fullerene-like hollow spheres, the metallasupramolecular chemistry of pentaphosphaferrocenes and CuBr2 afforded a conceptually new product, a compact 3.2 nm sized supramolecule [{1d}6(CuBr)32(CH3CN)6] formed by six largest pentaphosphaferrocene units [CpBIGFe(η5-P5)] (1d: CpBIG = η5-C5(4-nBuC6H4)5) so far and a framework of 32 copper and 32 bromide ions.

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR.

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes.

NMR derived changes of lipoprotein particle concentrations related to impaired fasting glucose, impaired glucose tolerance, or manifest type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2D) and corresponding borderline states, impaired fasting glucose (IFG) and/or glucose tolerance (IGT), are associated with dyslipoproteinemia. It is important to distinguish between factors that cause T2D and that are the direct result of T2D. METHODS: The lipoprotein subclass patterns of blood donors with IFG, IGT, with IFG combined with IGT, and T2D are analyzed by nuclear magnetic resonance (NMR) spectroscopy. The development of lipoprotein patterns with time is investigated by using samples retained for an average period of 6 years. In total 595 blood donors are classified by oral glucose tolerance test (oGTT) and their glycosylated hemoglobin (HbA1c) concentrations. Concentrations of lipoprotein particles of 15 different subclasses are analyzed in the 10,921 NMR spectra recorded under fasting and non-fasting conditions. The subjects are assumed healthy according to the strict regulations for blood donors before performing the oGTT. RESULTS: Under fasting conditions manifest T2D exhibits a significant concentration increase of the smallest HDL particles (HDL A) combined with a decrease in all other HDL subclasses. In contrast to other studies reviewed in this paper, a general concentration decrease of all LDL particles is observed that is most prominent for the smallest LDL particles (LDL A). Under normal nutritional conditions a large, significant increase of the concentrations of VLDL and chylomicrons is observed for all groups with IFG and/or IGT and most prominently for manifest T2D. As we show it is possible to obtain an estimate of the concentrations of the apolipoproteins Apo-A1, Apo-B100, and Apo-B48 from the NMR data. In the actual study cohort, under fasting conditions the concentrations of the lipoproteins are not increased significantly in T2D, under non-fasting conditions only Apo-B48 increases significantly. CONCLUSION: In contrast to other studies, in our cohort of "healthy" blood donors the T2D associated dyslipoproteinemia does not change the total concentrations of the lipoprotein particles produced in the liver under fasting and non-fasting conditions significantly but only their subclass distributions. Compared to the control group, under non-fasting conditions participants with IGT and IFG or T2D show a substantial increase of plasma concentrations of those lipoproteins that are produced in the intestinal tract. The intestinal insulin resistance becomes strongly observable.

Dynamics and determinants of human plasma bile acid profiles during dietary challenges.

In recent years, bile acids (BA) have received great interest due to their pleiotropic biological activity and the presence of plasma membrane-bound and nuclear receptors. Moreover, BA in blood have been identified by metabolite screening approaches as biomarkers that are associated with various diseases and even with a human longevity phenotype. With the growing interest in the microbiota contribution to the health-disease trajectory, BA that undergo deconjugation and other modifications by bacteria in the large intestine have become a prime target as a microbiome diversity modifier. We here profiled BA by a quantitative and a semiquantitative approach in 15 healthy and phenotypically very similar young individuals for over a 36-h fasting period, an oral glucose tolerance test (OGTT), and an oral lipid tolerance test (OLTT). We demonstrate a remarkable heterogeneity of the responses and describe the different dynamics of the plasma changes that likely originate from different routes by which BA enters the peripheral blood, and that may represent a direct secretion from the liver into the blood and a route that reaches the blood as a spill-over after passing from the gallbladder through the intestine and the portal system. We discuss the finding that an individual transport process involved in the passage of BA could be a critical determinant in the kinetics of plasma appearance and the overall phenotypic variability found.

Au-Containing Coordination Polymers Based on Polyphosphorus Ligand Complexes.

Whereas the self-assembly of pentaphosphaferrocenes [CpRFe(η5-P5)] (CpR = Cp*, Cp×, and CpBn) with Cu and Ag salts has been well-studied in the past, the coordination chemistry toward Au complexes has been left untouched so far. Herein, the results of the self-assembly processes of [CpRFe(η5-P5)] with Au salts of different anions (GaCl4-, SbF6-, and Al(OC(CF3)3)4 (TEF-)) are reported. Next to a variety of molecular coordination products, the first coordination polymers based on polyphosphorus ligand complexes and Au salts are also obtained. Thereby, a 2D coordination polymer comprising metal vacancies is isolated. In all products, the Au centers are coordinated in a linear or a trigonal planar environment. In solution, highly dynamic processes are observed. Variable-temperature NMR spectroscopy, solid-state NMR spectroscopy, and X-ray powder diffraction were applied to gain further insight into selected coordination compounds.

Reactivity of Cu(I) Nacnac Complexes Toward Polypnictogen Compounds.

The nacnac Cu(I) compound [LCu(MeCN)] (2) (L = [{N(C6H3Me2-2,6)C(Me)}2CH]-) was reacted with complexes containing aromatic cyclo-E5 ([Cp*Fe(η5-E5)], E = P (1a), As (1b), Cp* = η5-C5Me5), cyclo-P4 ([Cp‴Co(η4-P4)] (3), Cp‴ = η5-C5H2tBu3) and cyclo-E3 ligands ([Cp‴Ni(η3-E3)], E = P (4a), As (4b)) yielding the heterometallic complexes [(Cp*Fe)(µ,η5:2-E5)(LCu)] (E = P (5a), As (5b)), [(Cp*Fe)(µ3,η5:2:1-E5)(LCu)2] (E = P (6a), As (6b)), [(Cp‴Co)(µ,η4:2-P4)(LCu)] (7), [(Cp‴Co)(µ3,η4:2:1-P4)(LCu)2] (8), and [(Cp‴Ni)(µ,η3:2-E3)(LCu)] (E = P (9a), As (9b)). These complexes are rare examples of the coordination of a group 11 metal to aromatic cyclo-En (E = P, As; n = 3-5) ligands. All products were comprehensively characterized by crystallographic and spectroscopic methods. Their dynamic behavior in solution was studied by VT (variable-temperature) NMR spectroscopy, and their electronic structures were elucidated by DFT calculations.

Three-Component Self-Assembly Changes its Course: A Leap from Simple Polymers to 3D†Networks of Spherical Host-Guest Assemblies.

One-pot self-assembly reactions of the polyphosphorus complex [Cp*Fe(η5 -P5 )] (A), a coinage metal salt AgSbF6 , and flexible aliphatic dinitriles NC(CH2 )x CN (x=1-10) yield 1D, 2D, and 3D coordination polymers. The seven-membered backbone of the dinitrile was experimentally found as the borderline for the self-assembly system furnishing products of different kinds. At x<7, various rather simple polymers are exclusively formed possessing either 0D or 1D Ag/A structural motifs connected by dinitrile spacers, while at x≥7, the self-assembly switches to unprecedented extraordinary 3D networks of nano-sized host-guest assemblies (SbF6 )@[(A)9 Ag11 ]11+ (x=7) or (A)@[(A)12 Ag12 ]12+ (x=8-10) linked by dinitriles. The polycationic nodes represent the first superspheres based on A and silver and are host-guest able. All products are characterized by NMR spectroscopy, mass spectrometry, and single-crystal X-ray diffraction. The assemblies [(A)12 Ag12 ]12+ were visualized by transmission electron microscopy.

Correction to: A suite of 19F based relaxation dispersion experiments to assess biomolecular motions.

In the original publication, Figures 3 and 6 were displayed incorrectly due to a mistake made by the publisher. The correct version of Figs. 3 and 6 are given below.

RNA and DNA Binding Epitopes of the Cold Shock Protein TmCsp from the Hyperthermophile Thermotoga maritima.

Prokaryotic cold shock proteins (CSPs) are considered to play an important role in the transcriptional and translational regulation of gene expression, possibly by acting as transcription anti-terminators and "RNA chaperones". They bind with high affinity to single-stranded nucleic acids. Here we report the binding epitope of TmCsp from Thermotoga maritima for both single-stranded DNA and RNA, using heteronuclear 2D NMR spectroscopy. At "physiological" growth temperatures of TmCsp (≥ 343 K), all oligonucleotides studied have dissociation constants between 1.6 ((dT)7) and 25.2 ((dA)7) µM as determined by tryptophan fluorescence quenching. Reduction of the temperature to 303 K leads to a pronounced increase of affinity for thymidylate (dT)7 and uridylate (rU)7 heptamers with dissociation constants of 4.0 and 10.8 nM, respectively, whereas the weak binding of TmCsp to cytidylate, adenylate, and guanylate heptamers (dC)7, (dA)7, and (dT)7 is almost unaffected by temperature. The change of affinities of TmCsp for (dT)7 and (rU)7 by approximately 3 orders of magnitude shows that it represents a cold chock sensor that switches on the cold shock reaction of the cell. A temperature dependent conformational switch of the protein is required for this action. The binding epitope on TmCsp for the ssDNA and RNA heptamers is very similar and comprises ß-strands 1 and 2, the loop ß1-ß2 as well as the loops connecting ß3 with ß4 and ß4 with ß5. Besides the loop regions, surprisingly, mainly the RNA-binding motif RNP1 is involved in ssDNA and RNA binding, while only two amino acids, H28 and W29, of the postulated RNA-binding motif RNP2 interact with the uridylate and thymidylate homonucleotides, although a high affinity in the nanomolar range is achieved. This is in contrast to the binding properties of other CSPs or cold shock domains, where RNP1 as well as RNP2 are involved in binding. TmCsp takes up a unique position since it is the only one which possesses a tryptophan residue instead of a usually highly conserved phenylalanine or tyrosine residue at the end of RNP2. NMR titrations suggest that neither (dT)7 nor (rU)7 represent the full binding motif and that non-optimal intercalation of W29 into these oligonucleotides blocks the access of the RNP2 site to the DNA or RNA. NMR-experiments with (dA)7 suggest an interaction of W29 with the adenine ring. Full binding seems to require at least one single purine base well-positioned within a thymine- or uracil-rich stretch of nucleic acids.

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions.

Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions.

Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of 1H and 15N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficients B1 and B2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1H and 15N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For Hα and HN correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients.

Iodination of cyclo-E5 -Complexes (E=P, As).

In a high-yield one-pot synthesis, the reactions of [Cp*M(η5 -P5 )] (M=Fe (1), Ru (2)) with I2 resulted in the selective formation of [Cp*MP6 I6 ]+ salts (3, 4). The products comprise unprecedented all-cis tripodal triphosphino-cyclotriphosphine ligands. The iodination of [Cp*Fe(η5 -As5 )] (6) gave, in addition to [Fe(CH3 CN)6 ]2+ salts of the rare [As6 I8 ]2- (in 7) and [As4 I14 ]2- (in 8) anions, the first di-cationic Fe-As triple decker complex [(Cp*Fe)2 (µ,η5:5 -As5 )][As6 I8 ] (9). In contrast, the iodination of [Cp*Ru(η5 -As5 )] (10) did not result in the full cleavage of the M-As bonds. Instead, a number of dinuclear complexes were obtained: [(Cp*Ru)2 (µ,η5:5 -As5 )][As6 I8 ]0.5 (11) represents the first Ru-As5 triple decker complex, thus completing the series of monocationic complexes [(CpR M)2 (µ,η5:5 -E5 )]+ (M=Fe, Ru; E=P, As). [(Cp*Ru)2 As8 I6 ] (12) crystallizes as a racemic mixture of both enantiomers, while [(Cp*Ru)2 As4 I4 ] (13) crystallizes as a symmetric and an asymmetric isomer and features a unique tetramer of {AsI} arsinidene units as a middle deck.

Complete sequential assignment and secondary structure prediction of the cannulae forming protein CanA from the hyperthermophilic archaeon Pyrodictium abyssi.

CanA from Pyrodictium abyssi forms a heat-resistant organic hollow-fiber network together with CanB and CanC. An N-terminally truncated construct of CanA (K1-CanA) gave NMR spectra of good quality that could be assigned by three-dimensional NMR methods on 15N and 13C-15N enriched protein. We assigned the chemical shifts of 96% of all backbone 1HN atoms, 98% of all backbone 15N atoms, 100% of all 13Cα atoms, 100% of all 1Hα atoms, 90% of all 13C' atoms, and 100% of the 13Cß atoms. Two short helices and 10 ß-strands are estimated from an analysis of the chemical shifts leading to a secondary structure content of K1-CanA of 6% helices, 44% ß-pleated sheets, and 50% coils.

Pressure-dependent electronic structure calculations using integral equation-based solvation models.

Recent methodological progress in quantum-chemical calculations using the "embedded cluster reference interaction site model" (EC-RISM) integral equation theory is reviewed in the context of applying it as a solvation model for calculating pressure-dependent thermodynamic and spectroscopic properties of molecules immersed in water. The methodology is based on self-consistent calculations of electronic and solvation structure around dissolved molecules where pressure enters the equations via an appropriately chosen solvent response function and the pure solvent density. Besides specification of a dispersion-repulsion force field for solute-solvent interactions, the EC-RISM approach derives the electrostatic interaction contributions directly from the wave function. We further develop and apply the method to a variety of benchmark cases for which computational or experimental reference data are either available in the literature or are generated specifically for this purpose in this work. Starting with an enhancement to predict hydration free energies at non-ambient pressures, which is the basis for pressure-dependent molecular population estimation, we demonstrate the performance on the calculation of the autoionization constant of water. Spectroscopic problems are addressed by studying the biologically relevant small osmolyte TMAO (trimethylamine N-oxide). Pressure-dependent NMR shifts are predicted and compared to experiments taking into account proper computational referencing methods that extend earlier work. The experimentally observed IR blue-shifts of certain vibrational bands of TMAO as well as of the cyanide anion are reproduced by novel methodology that allows for weighing equilibrium and non-equilibrium solvent relaxation effects. Taken together, the model systems investigated allow for an assessment of the reliability of the EC-RISM approach for studying pressure-dependent biophysical processes.

High pressure response of 1H NMR chemical shifts of purine nucleotides.

The study of the pressure response by NMR spectroscopy provides information on the thermodynamics of conformational equilibria in proteins and nucleic acids. For obtaining a database for expected pressure effects on free nucleotides and nucleotides bound in macromolecular complexes, the pressure response of 1H chemical shifts and J-coupling constants of the purine 5'-ribonucleotides AMP, ADP, ATP, GMP, GDP, and GTP were studied in the absence and presence of Mg2+-ions. Experiments are supported by quantum-chemical calculations of populations and chemical shift differences in order to corroborate structural interpretations and to estimate missing data for AMP. The preference of the ribose S puckering obtained from the analysis of the experimental J-couplings is also confirmed by the calculations. In addition, the pressure response of the non-hydrolysable GTP analogues GppNHp, GppCH2p, and GTPγS was examined within a pressure range up to 200 MPa. As observed earlier for 31P NMR chemical shifts of these nucleotides the pressure dependence of chemical shifts is clearly non-linear in most cases. In di- and tri-phospho nucleosides, the resonances of the two protons bound to the ribose 5' carbon are non-equivalent and can be observed separately. The gg-rotamer at C4'- C5' bond is strongly preferred and the downfield shifted resonance can be assigned to the H5″ proton in the nucleotides. In contrast, in adenosine itself the frequencies of the two resonances are interchanged.

The pressure and temperature perturbation approach reveals a whole variety of conformational substates of amyloidogenic hIAPP monitored by 2D NMR spectroscopy.

The intrinsically disordered human islet amyloid polypeptide (hIAPP) is a 37 amino acid peptide hormone that is secreted by pancreatic beta cells along with glucagon and insulin. The glucose metabolism of humans is regulated by a balanced ratio of insulin and hIAPP. The disturbance of this balance can result in the development of type-2 diabetes mellitus (T2DM), whose pathogeny is associated by self-assembly induced aggregation and amyloid deposits of hIAPP into nanofibrils. Here, we report pressure- and temperature-induced changes of NMR chemical shifts of monomeric hIAPP in bulk solution to elucidate the contribution of conformational substates in a residue-specific manner in their role as molecular determinants for the initial self-assembly. The comparison with a similar peptide, the Alzheimer peptide Aß(1-40), which is leading to self-assembly induced aggregation and amyloid deposits as well, reveals that in both peptides highly homologous areas exist (Q10-|L16 and N21-L27 in hIAPP and Q15-A21 and S26-I32 in Aß). The N-terminal area of hIAPP around amino acid residues 3-20 displays large differences in pressure sensitivity compared to Aß, pinpointing to a different structural ensemble in this sequence element which is of helical origin in hIAPP. Knowledge of the structural nature of the highly amyloidogenic hIAPP and the differences with respect to the conformational ensemble of Aß(1-40) will help to identify molecular determinants of self-assembly as well as cross-seeded assembly initiated aggregation and help facilitate the rational design of drugs for therapeutic use.

1H NMR spectroscopy quantifies visibility of lipoproteins, subclasses, and lipids at varied temperatures and pressures.

NMR-based quantification of human lipoprotein (sub)classes is a powerful high-throughput method for medical diagnostics. We evaluated select proton NMR signals of serum lipoproteins for elucidating the physicochemical features and the absolute NMR visibility of their lipids. We separated human lipoproteins of different subclasses by ultracentrifugation and analyzed them by 1H NMR spectroscopy at different temperatures (283-323 K) and pressures (0.1-200 MPa). In parallel, we determined the total lipid content by extraction with chloroform/methanol. The visibility of different lipids in the 1H NMR spectra strongly depends on temperature and pressure: it increases with increasing temperatures but decreases with increasing pressures. Even at 313 K, only part of the lipoprotein is detected quantitatively. In LDL and in HDL subclasses HDL2 and HDL3, only 39%, 62%, and 90% of the total cholesterol and only 73%, 70%, and 87% of the FAs are detected, respectively. The choline head groups show visibilities of 43%, 75%, and 87% for LDL, HDL2, and HDL3, respectively. The description of the NMR visibility of lipid signals requires a minimum model of three different compartments, A, B, and C. The thermodynamic analysis of compartment B leads to melting temperatures between 282 K and 308 K and to enthalpy differences that vary for the different lipoproteins as well as for the reporter groups selected. In summary, we describe differences in NMR visibility of lipoproteins and variations in biophysical responses of functional groups that are crucial for the accuracy of absolute NMR quantification.

From nano-balls to nano-bowls.

Pentaphosphaferrocene [Cp*Fe(η5-P5)] in combination with Cu(i) halides is capable of a template-directed synthesis of fullerene-like spheres. Herein, we present the use of a triple decker complex as template that leads to the formation of unprecedented 'nano-bowls'. These spherical domes resemble the truncated fullerenes I h-C80 and represent a novel spherical arrangement in the chemistry of spherical molecules.