Use of EDTA to minimize ionic strength dependent frequency shifts in the <Superscript>1</Superscript>H NMR spectra of urine

The ¹H NMR spectrum of urine exhibits a large number of detectable metabolites and is, therefore, highly suitable for the study of perturbations caused by disease, toxicity, nutrition or environmental factors in humans and animals. However, variations in the chemical shifts and intensities due to altered pH and ionic strength present a challenge in NMR-based studies. With a view towards understanding and minimizing the effects of these variations, we have extensively studied the effects of ionic strength and pH on the chemical shifts of common urine metabolites and their possible reduction using EDTA (ethylenediaminetetraacetic acid). ¹H NMR chemical shifts for alanine, citrate, creatinine, dimethylamine, glycine, histidine, hippurate, formate and the internal reference, TSP (trimethylsilylpropionic acid-d₄, sodium salt) obtained under different conditions were used to assess each effect individually. EDTA minimizes the frequency shifts of the metabolites that have a propensity for metal binding. Chelation of such metal ions is evident from the appearance of signals from EDTA complexed to divalent metal ions such as calcium and magnesium. Not surprisingly, increasing the buffer concentration or buffer volume also minimizes pH dependent frequency shifts. The combination of EDTA and an appropriate buffer effectively minimizes both pH dependent frequency shifts and ionic strength dependent intensity variations in urine NMR spectra. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The 13C chemical shifts of amino acids in aqueous solution containing organic solvents: Application to the secondary structure characterization of peptides in aqueous trifluoroethanol solution

Summary The ¹³C chemical shifts for all of the protonated carbons of the 20 common amino acid residues in the protected linear pentapeptide Gly-Gly-X-Gly-Gly have been obtained in water at low pH as well as in aqueous solution containing 10, 20 and 30% acetonitrile or trifluoroethanol. Dioxane was used as an internal reference and its carbon chemical shift value was found to be 66.6 ppm relative to external TMS in water. Comparison of the different referencing methods for ¹³C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate. In the present study, external TSP was chosen to define the 0 ppm of the ¹³C chemical shift scale. When the difference in referencing the dioxane carbon resonance is taken into account, the carbon chemical shift values of the amino acids in aqueous solution are similar to those previously reported (Richarz and Wüthrich (1978) Biopolymers, 17, 2133–2141; Howarth and Lilley (1979) Prog. NMR Spectrosc., 12, 1–40). The pentapeptides studied were assumed to be in a random coil conformation and the measured ¹³C chemical shifts were used as reference values to correlate carbon chemical shifts with the secondary structure of two well-characterized peptides, bombesin and the 1–29 amino acid fragment of Nle²⁷ human growth hormone-releasing factor. In both cases, the C chemical shifts exhibited a characteristic positive deviation from the random coil values, which indicates the presence of -helices.     

Molecular dynamics simulations of HPr under hydrostatic pressure

The histidine-containing protein (HPr) plays an important role in the phosphotransferase system (PTS). The deformations induced on the protein structure at high hydrostatic pressure values (4, 50, 100, 150, and 200 MPa) were previously (H. Kalbitzer, A. G?rler, H. Li, P. Dubovskii, A. Hengstenberg, C. Kowolik, H. Yamada, and K. Akasaka, Protein Science 2000, Vol. 9, pp. 693-703) analyzed by NMR experiments: the nonlinear variations of the amide chemical shifts at high pressure values were supposed to arise from induced shifts in the protein conformational equilibrium. Molecular dynamics (MD) simulations are here performed, to analyze the protein internal mobility at 0.1 MPa, and to relate the nonlinear variations of chemical shifts observed at high pressure, to variations in conformational equilibrium. The global features of the protein structure are only slightly modified along the pressure. Nevertheless, the values of the Voronoi residues volumes show that the residues of alpha-helices are more compressed that those belonging to the beta-sheet. The alpha-helices are also displaying the largest internal mobility and deformation in the simulations. The nonlinearity of the 1H chemical shifts, computed from the MD simulation snapshots, is in qualitative agreement with the nonlinearity of the experimentally observed chemical shifts.     

Interaction of tryptophan containing oligopeptide with d-CpGpCpG by proton NMR

The binding of tetrapeptide Lys-Trp-Gly-Lys OtBu to d-CpGpCpG has been studied by proton NMR at 90 MHz and 400 MHz. Changes in chemical shift have been observed in the temperature range 275-335 K. Interaction with tetrapeptide Lys-Ala-Ala-Lys NHEt has been studied in order to ascertain the contribution to changes in chemical shift due to the electrostatic interactions alone. On addition of Lys-Trp-Gly-Lys OtBu to d-CGCG, the H-5 and H-6 resonances of internal cytosine shift upfield about 0.04-0.07 ppm at 275 K. The upfield shift in external Cytosine are relatively small about 0.01 ppm. Changes in chemical shifts of internal and external Guanine (H-8) are indistinguishable being in the range 0.02-0.11 ppm. The changes in chemical shift of Tryptophan ring protons on binding to oligonucleotide are considerably large, it being typically an upfield shift to 0.18-0.53 ppm at 275 K. The changes in chemical shift of all resonances decrease with temperature. The observations suggest intercalation of Tryptophan ring in d-CGCG. Using the magnetic anisotropy ring current shifts, overlap geometries of Tryptophan ring in d(C-G) and d(G-C) sites of d-CGCG have been proposed. The same has been verified by using Corey-Pauling-Koltun models.     

Internal pH indicators for biomolecular NMR

We describe a non-invasive technique for determining pH in biomolecular NMR sample using buffer components (formate, tris, piperazine, and imidazole) as internal pH indicators, whose (1)H NMR chemical shifts are sensitive to pH in a range from 2.5 to 9.8. This method is suitable for a wide range of applications where samples are handled intensively during NMR titrations or in high throughput analysis in structural genomics or metabolomics.     

Models for biological ion exchangers. I. Proton magnetic resonance studies of water structure in Bio-Rex 70.

Bio-Rex 70, a carboxylic acid cation exchanger, is studied as a biological ion-exchanger resin model for cellular cytoplasm. High-resolution proton magnetic resonance spectra of 1l ionic forms of Bio-Rex 70 are determined. From measured cation exchange capacities, water contents, and chemical shifts for the resin-phase water protons, the dependence of the chemical shift on the counter ion is calculated. The observed chemical shifts (Hz/geq/kg internal water, referred to the Ba2+ form) for each ionic form are: H+, --0.5; Li+, --0.2; Na+, 1.2; K+, 2; Rb+, 2.1; Ag+, --0.4; NH4+, --2.0; NMe4+, 1.2; NEt4+, 1.8; Mg2+, --2.0; Ca2+, --1.5; Sr2+, --0.6; Ba2+, 0.0 Zn2+, --2.3; Cd2+, --4.7; and La3+, --3.3. The results are in good agreement with earlier studies on Dowex 50, indicating that the carboxylate ion exchanger behaves like a concentrated polyelectrolyte. The widths at half-height for the internal water peaks of the polyvalent forms are quite large, ranging from 40 to 100 Hz.     

Ring current effects in the conformation dependent NMR chemical shifts of aliphatic protons in the basic pancreatic trypsin inhibitor.

Previously, a highly refined crystal structure and energy refined atomic coordinates were obtained for the basic pancreatic trypsin inhibitor, as well as numerous individual resonance assignments in the 1H NMR spectrum. These data were now used to investigate the contributions from the local ring current fields of the aromatic rings to the overall conformation dependent chemical shifts in this globular protein. A program was written which allowed the consideration of certain aspects of internal mobility of the protein, and the different commonly used ring current equa tions were compared. These studies indicate that ring current shifts are the dominant contribution to the observed conformation dependent chemical shifts of the peripheral aliphatic side chain protons. On the other hand, it appears that ring current shifts do not make dominant contributions to the conformation dependent shifts of the backbone alpha- and amide protons or the aromatic protons in the inhibitor. On the basis of the empirical calibration with the peripheral aliphatic side chain protons, the Johnson-Bovey ring current equation was selected for an analysis of the ring geometries of two prolines in the inhibitor.     

Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated (13)C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (13)CO experiments however the large separation between (13)CO and (13)C(alpha) chemical shifts offers hope that robust (13)CO dispersion profiles can be recorded on uniformly (13)C labeled samples, leading to the extraction of accurate (13)CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-(13)C]-pyruvate and NaH(13)CO(3,) or uniformly labeled, generated from (13)C-glucose. Very similar (13)CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.     

An automated system designed for large scale NMR data deposition and annotation: application to over 600 assigned chemical shift data entries to the BioMagResBank from the Riken Structural Genomics/Proteomics Initiative internal database

Biomolecular NMR chemical shift data are key information for the functional analysis of biomolecules and the development of new techniques for NMR studies utilizing chemical shift statistical information. Structural genomics projects are major contributors to the accumulation of protein chemical shift information. The management of the large quantities of NMR data generated by each project in a local database and the transfer of the data to the public databases are still formidable tasks because of the complicated nature of NMR data. Here we report an automated and efficient system developed for the deposition and annotation of a large number of data sets including (1)H, (13)C and (15)N resonance assignments used for the structure determination of proteins. We have demonstrated the feasibility of our system by applying it to over 600 entries from the internal database generated by the RIKEN Structural Genomics/Proteomics Initiative (RSGI) to the public database, BioMagResBank (BMRB). We have assessed the quality of the deposited chemical shifts by comparing them with those predicted from the PDB coordinate entry for the corresponding protein. The same comparison for other matched BMRB/PDB entries deposited from 2001-2011 has been carried out and the results suggest that the RSGI entries greatly improved the quality of the BMRB database. Since the entries include chemical shifts acquired under strikingly similar experimental conditions, these NMR data can be expected to be a promising resource to improve current technologies as well as to develop new NMR methods for protein studies.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.     

Relationship between nuclear magnetic resonance chemical shift and protein secondary structure

An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships. Similar studies, performed on smaller databases, for 13C and 15N chemical shifts reveal equally strong correlations to protein secondary structure. Among the more interesting results to emerge from this work is the finding that all 20 naturally occurring amino acids experience a mean alpha-1H upfield shift of 0.39 parts per million (from the random coil value) when placed in a helical configuration. In a like manner, the alpha-1H chemical shift is found to move downfield by an average of 0.37 parts per million when the residue is placed in a beta-strand or extended configuration. Similar changes are also found for amide 1H, carbonyl 13C, alpha-13C and amide 15N chemical shifts. Other relationships between chemical shift and protein conformation are also uncovered; in particular, a correlation between helix dipole effects and amide proton chemical shifts as well as a relationship between alpha-proton chemical shifts and main-chain flexibility. Additionally, useful relationships between alpha-proton chemical shifts and backbone dihedral angles as well as correlations between amide proton chemical shifts and hydrogen bond effects are demonstrated.     

Quantum mechanical calculations and experimental measurement of N-terminal charge effects on 1HN and 1HCα chemical shifts in peptides

Nuclear magnetic resonance spectroscopists are increasingly utilizing chemical shifts to characterize the secondary structure of proteins. The present study addresses the effects that the positively charged amino group at the N-terminus of a peptide has on ¹HN and ¹HCα chemical shifts along the chain. This information is necessary for interpreting chemical shift data for proteins and/or for peptides that are used as models for protein structure. The chemical shifts for the ¹H resonances of four peptides that differ only in the location of their N-terminii are assigned using two-dimensional nmr spectroscopy. The peptides have sequences derived from the β subunit of the glycoprotein hormone human chorionic gonadotropin (hCG-β). Comparison of the ¹HN and the ¹HCα chemical shifts for residues common to all four peptides reveals downfield shifts for ¹HN and the ¹HCα resonances within three residues of the N-terminus compared with chemical shifts in the interior of the peptide. The magnitude of the downfield shift is larger for resonances nearer the N-terminus. Quantum mechanical calculations of the ¹HN and ¹HCα chemical shifts in peptides constructed with six alanine units also predict a significant terminus effect. The calculations agree both qualitatively and quantitatively with the experimental data. The inductive nature of the end effect is confirmed in the calculations by Mulliken population analysis. End effects should be taken into account in determining protein secondary structures from chemical shifts. © 1996 John Wiley & Sons, Inc.     

Two-dimensional magic angle spinning NMR investigation of naturally occurring chitins: precise 1H and 13C resonance assignment of alpha- and beta-chitin

13C homonuclear through-bond correlations of alpha- and beta-chitin were determined by using two-dimensional (2D) INADEQUATE spectra of these allomorphs purified from crab shell and squid pen, respectively. The 2D (13)C-(13)C correlation spectra where two directly bonded carbons share a common double-quantum frequency (DQ) enabled us to precisely assign all (13)C resonances of the chitin allomorphs for the first time. Following the complete (13)C assignment, (1)H chemical shifts of protons attached to each carbon nuclei were assigned by 2D frequency-switched Lee-Goldberg (FSLG) (1)H-(13)C heteronuclear correlation (HETCOR) spectra of the chitin allomorphs, recorded with a short mixing time (60 micros) to provide isotropic (1)H-(13)C chemical shift correlations between bonded pairs proton and carbon nuclei. From the (13)C and (1)H chemical shifts of chitin allomorphs, all 2-deoxy-2-acetamide-D-glucose (N-acetyl-D-glucosamine) monomer units in each allomorph were revealed to be an identical (13)C-(13)C backbone conformation and magnetically equivalent. In addition, it was strongly suggested that there are two different hydrogen-bonding patterns at the hydroxyl groups of alpha-chitin by comparing (1)H chemical shifts at the C6 site of alpha-chitin with those at the same site of beta-chitin.     

A probabilistic model for secondary structure prediction from protein chemical shifts

Protein chemical shifts encode detailed structural information that is difficult and computationally costly to describe at a fundamental level. Statistical and machine learning approaches have been used to infer correlations between chemical shifts and secondary structure from experimental chemical shifts. These methods range from simple statistics such as the chemical shift index to complex methods using neural networks. Notwithstanding their higher accuracy, more complex approaches tend to obscure the relationship between secondary structure and chemical shift and often involve many parameters that need to be trained. We present hidden Markov models (HMMs) with Gaussian emission probabilities to model the dependence between protein chemical shifts and secondary structure. The continuous emission probabilities are modeled as conditional probabilities for a given amino acid and secondary structure type. Using these distributions as outputs of first‐ and second‐order HMMs, we achieve a prediction accuracy of 82.3%, which is competitive with existing methods for predicting secondary structure from protein chemical shifts. Incorporation of sequence‐based secondary structure prediction into our HMM improves the prediction accuracy to 84.0%. Our findings suggest that an HMM with correlated Gaussian distributions conditioned on the secondary structure provides an adequate generative model of chemical shifts. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Nitrogen-15 chemical shifts in AT (adenine-thymine) and CG (cytosine-guanine) nucleic acid base pairs

This paper presents ab initio (DFT) calculations of the 15N chemical shifts in AT (Adenine-Thymine) and CG (Cytosine-Guanine) nucleic acid base pairs. Calculations were done on 14 AT and 18 CG base pairs using experimental (X-ray) geometries obtained from several DNA decamers. The calculated chemical shifts are compared with the experimental values in the pure bases and subjected to statistical analysis to explore their sensitivity to the local geometry and pair helix parameters. The results indicate that the 15N chemical shifts, isotropic and principal components are quite sensitive to small changes in the geometry of the pairs, but they do not correlate well with the helix pair parameters. From the statistical analysis, several linear correlations between structural parameters and chemical shifts emerge. These relationships may serve as a foundation to extract information on molecular structure from 15N chemical shift measurements.     

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects

Summary In this study we report on the ¹H, ¹³C and ¹⁵N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of ¹H, ¹³C and ¹⁵N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.     

Predicting 15N chemical shifts in proteins using the preceding residue-specific individual shielding surfaces from φ, ψi−1, and χ1torsion angles

Empirical shielding surfaces are most commonly used to predict chemical shifts in proteins from known backbone torsion angles, phi and psi. However, the prediction of (15)N chemical shifts using this technique is significantly poorer, compared to that for the other nuclei such as (1)H(alpha), (13)C(alpha), and (13)C(beta). In this study, we investigated the effects from the preceding residue and the side-chain geometry, chi(1), on (15)N chemical shifts by statistical methods. For an amino acid sequence XY, the (15)N chemical shift of Y is expressed as a function of the amino acid types of X and Y, as well as the backbone torsion angles, phi and psi(i-1). Accordingly, 380 empirical 'Preceding Residue Specific Individual (PRSI)' (15)N chemical shift shielding surfaces, representing all the combinations of X and Y (except for Y=Pro), were built and used to predict (15)N chemical shift from phi and psi(i-1). We further investigated the chi(1) effects, which were found to account for differences in (15)N chemical shifts by approximately 5 ppm for amino acids Val, Ile, Thr, Phe, His, Tyr, and Trp. Taking the chi(1) effects into account, the chi(1)-calibrated PRSI shielding surfaces (XPRSI) were built and used to predict (15)N chemical shifts for these amino acids. We demonstrated that (15)N chemical shift predictions are significantly improved by incorporating the preceding residue and chi(1) effects. The present PRSI and XPRSI shielding surfaces were extensively compared with three recently published programs, SHIFTX (Neal et al., 2003), SHIFTS (Xu and Case, 2001 and 2002), and PROSHIFT (Meiler, 2003) on a set of ten randomly selected proteins. A set of Java programs using XPRSI shielding surfaces to predict (15)N chemical shifts in proteins were developed and are freely available for academic users at http://www.pronmr.com or by sending email to one of the authors Yunjun Wang (yunjunwang@yahoo.com).     

Toward the quantum chemical calculation of nuclear magnetic resonance chemical shifts of proteins

Despite the many protein structures solved successfully by nuclear magnetic resonance (NMR) spectroscopy, quality control of NMR structures is still by far not as well established and standardized as in crystallography. Therefore, there is still the need for new, independent, and unbiased evaluation tools to identify problematic parts and in the best case also to give guidelines that how to fix them. We present here, quantum chemical calculations of NMR chemical shifts for many proteins based on our fragment-based quantum chemical method: the adjustable density matrix assembler (ADMA). These results show that (13)C chemical shifts of reasonable accuracy can be obtained that can already provide a powerful measure for the structure validation. (1)H and even more (15)N chemical shifts deviate more strongly from experiment due to the insufficient treatment of solvent effects and conformational averaging.     

1H-nmr study of protected methionine homo-oligopeptides in helix-supporting environment

¹H-nmr spectra for a series of Boc-L -(Met)_n-OMe (n = 2–9) homo-oligopeptides have been observed in the helix-supporting solvent trifluoroethanol (TFE) at millimolar concentrations. Interfering solvent peaks were eliminated using two decoupling frequencies to selectively remove the methylene and hydroxyl protons of the solvent. Comparisons with specifically α-deuterated homo-oligopeptides gave complete assignments of the NH region of the Boc-Met_n-OMe oligomers up to the heptapeptide. Analysis of chemical shifts, coupling constants, and temperature dependence of chemical shifts suggests that up to the hexapeptide, similar structures exist in deuterochloroform and TFE. In contrast, nmr parameters at the heptapeptide for several internal residues differ in these solvents. These results suggest that a C₇ to α-helix transition may occur in TFE as the chain length of the methionine oligopeptides increases.