A simple method to adjust inconsistently referenced <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of proteins

Inconsistent ¹³C and ¹⁵N chemical shift referencing is a continuing problem associated with protein chemical shift assignments deposited in BioMagResBank (BMRB). Here we describe a simple and robust approach that can quantitatively determine the ¹³C and ¹⁵N referencing offsets solely from chemical shift assignment data and independently of 3D coordinate data. This novel structure-independent approach permitted the assessment and determination of ¹³C and ¹⁵N reference offsets for all protein entries deposited in the BMRB. Tests on 452 proteins with known 3D structures show that this structure-independent approach yields ¹³C and ¹⁵N referencing offsets that exhibit excellent agreement with those calculated on the basis of 3D structures. Furthermore, this protocol appears to improve the accuracy of chemical shift-derived secondary structural identification, and has been formally incorporated into a computer program called PSSI (http//www.pronmr.com).Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7441-3     

Linear analysis of carbon-13 chemical shift differences and its application to the detection and correction of errors in referencing and spin system identifications

Statistical analysis reveals that the set of differences between the secondary shifts of the α- and β-carbons for residues i of a protein (Δδ¹³C^α_i- Δδ¹³C^β_i) provides the means to detect and correct referencing errors for ¹H and ¹³C nuclei within a given dataset. In a correctly referenced protein dataset, linear regression plots of Δδ¹³C^α_i,Δδ¹³C^β_i, or Δδ¹H^α_i vs. (Δδ¹³C^α_i- Δδ¹³C^β_i) pass through the origin from two directions, the helix-to-coil and strand-to-coil directions. Thus, linear analysis of chemical shifts (LACS) can be used to detect referencing errors and to recalibrate the ¹H and ¹³C chemical shift scales if needed. The analysis requires only that the signals be identified with distinct residue types (intra-residue spin systems). LACS allows errors in calibration to be detected and corrected in advance of sequence-specific assignments and secondary structure determinations. Signals that do not fit the linear model (outliers) deserve scrutiny since they could represent errors in identifying signals with a particular residue, or interesting features such as a cis-peptide bond. LACS provides the basis for the automated detection of such features and for testing reassignment hypotheses. Early detection and correction of errors in referencing and spin system identifications can improve the speed and accuracy of chemical shift assignments and secondary structure determinations. We have used LACS to create a database of offset-corrected chemical shifts corresponding to nearly 1800 BMRB entries: 300 with and 1500 without corresponding three-dimensional (3D) structures. This database can serve as a resource for future analysis of the effects of amino acid sequence and protein secondary and tertiary structure on NMR chemical shifts.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-1717-0     

Pressure-dependent <Superscript>13</Superscript>C chemical shifts in proteins: origins and applications

Pressure-dependent ¹³C chemical shifts have been measured for aliphatic carbons in barnase and Protein G. Up to 200 MPa (2 kbar), most shift changes are linear, demonstrating pressure-independent compressibilities. CH₃, CH₂ and CH carbon shifts change on average by +0.23, −0.09 and −0.18 ppm, respectively, due to a combination of bond shortening and changes in bond angles, the latter matching one explanation for the γ-gauche effect. In addition, there is a residue-specific component, arising from both local compression and conformational change. To assess the relative magnitudes of these effects, residue-specific shift changes for protein G were converted into structural restraints and used to calculate the change in structure with pressure, using a genetic algorithm to convert shift changes into dihedral angle restraints. The results demonstrate that residual ¹³Cα shifts are dominated by dihedral angle changes and can be used to calculate structural change, whereas ¹³Cβ shifts retain significant dependence on local compression, making them less useful as structural restraints.     

Deuterium isotope effects on <Superscript>15</Superscript>N backbone chemical shifts in proteins

Quantum mechanical calculations are presented that predict that one-bond deuterium isotope effects on the ¹⁵N chemical shift of backbone amides of proteins, ¹Δ¹⁵N(D), are sensitive to backbone conformation and hydrogen bonding. A quantitative empirical model for ¹Δ¹⁵N(D) including the backbone dihedral angles, Φ and Ψ, and the hydrogen bonding geometry is presented for glycine and amino acid residues with aliphatic side chains. The effect of hydrogen bonding is rationalized in part as an electric-field effect on the first derivative of the nuclear shielding with respect to N–H bond length. Another contributing factor is the effect of increased anharmonicity of the N–H stretching vibrational state upon hydrogen bonding, which results in an altered N–H/N–D equilibrium bond length ratio. The N–H stretching anharmonicity contribution falls off with the cosine of the N–H···O bond angle. For residues with uncharged side chains a very good prediction of isotope effects can be made. Thus, for proteins with known secondary structures, ¹Δ¹⁵N(D) can provide insights into hydrogen bonding geometries. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conformational signatures of <Superscript>13</Superscript>C chemical shifts in RNA ribose

The conformational dependence of ¹³C chemical shift values of RNA riboses determined by liquid-state NMR spectroscopy was evaluated using data deposited for RNA structures in the RCSD and BMRB data bases. Results derived support the applicability of the canonical coordinates approach of Rossi and Harbison (J Magn Reson 151:1–8, 2001) in liquid-state NMR to assess the sugar pucker of ribose units in RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of mammalian Y145Stop prion protein amyloid fibrils

The Y145Stop prion protein (PrP23-144), which has been linked to the development of a heritable prionopathy in humans, is a valuable in vitro model for elucidating the structural and molecular basis of amyloid seeding specificities. Here we report the sequential backbone and side-chain ¹³C and ¹⁵N assignments of mouse and Syrian hamster PrP23-144 amyloid fibrils determined by using 2D and 3D magic-angle spinning solid-state NMR. The assigned chemical shifts were used to predict the secondary structures for the core regions of the mouse and Syrian hamster PrP23-144 amyloids, and the results compared to those for human PrP23-144 amyloid, which has previously been analyzed by solid-state NMR techniques.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak

Ahnak is a ~?700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca²⁺ signaling in cardiomyocytes by interacting with the β subunit of the L-type Ca²⁺ channel (Ca_V1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete ¹H, ¹³C and ¹⁵N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the Ca_V1.2-Ahnak complex.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB₅ toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM₁-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the ¹H, ¹³C, ¹⁵N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on ¹³C′, ¹³C^{α, 13}C^{β, 1}H^N and ¹⁵N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM₁-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens.     

Rapid and accurate calculation of protein 1H, 13C and 15N chemical shifts

A computer program (SHIFTX) is described which rapidly and accurately calculates the diamagnetic ¹H, ¹³C and ¹⁵N chemical shifts of both backbone and sidechain atoms in proteins. The program uses a hybrid predictive approach that employs pre-calculated, empirically derived chemical shift hypersurfaces in combination with classical or semi-classical equations (for ring current, electric field, hydrogen bond and solvent effects) to calculate ¹H, ¹³C and ¹⁵N chemical shifts from atomic coordinates. The chemical shift hypersurfaces capture dihedral angle, sidechain orientation, secondary structure and nearest neighbor effects that cannot easily be translated to analytical formulae or predicted via classical means. The chemical shift hypersurfaces were generated using a database of IUPAC-referenced protein chemical shifts – RefDB (Zhang et al., 2003), and a corresponding set of high resolution (<2.1 Å) X-ray structures. Data mining techniques were used to extract the largest pairwise contributors (from a list of 20 derived geometric, sequential and structural parameters) to generate the necessary hypersurfaces. SHIFTX is rapid (< 1 CPU second for a complete shift calculation of 100 residues) and accurate. Overall, the program was able to attain a correlation coefficient (r) between observed and calculated shifts of 0.911 (¹H), 0.980 (¹³C), 0.996 (¹³C), 0.863 (¹³CO), 0.909 (¹⁵N), 0.741 (¹HN), and 0.907 (sidechain ¹H) with RMS errors of 0.23, 0.98, 1.10, 1.16, 2.43, 0.49, and 0.30 ppm, respectively on test data sets. We further show that the agreement between observed and SHIFTX calculated chemical shifts can be an extremely sensitive measure of the quality of protein structures. Our results suggest that if NMR-derived structures could be refined using heteronuclear chemical shifts calculated by SHIFTX, their precision could approach that of the highest resolution X-ray structures. SHIFTX is freely available as a web server at http://redpoll.pharmacy.ualberta.ca.     

Measurement of protein backbone <Superscript>13</Superscript>CO and <Superscript>15</Superscript>N relaxation dispersion at high resolution

The ³¹P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the ³¹P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH₂p and GTPγS was measured in the absence and presence of Mg²⁺-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B ₁ was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg²⁺·GMP and Mg²⁺·GppNHp the second order pressure coefficients are positive. To describe the data of Mg²⁺·GppCH₂p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg²⁺ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure ³¹P NMR data on free Mg²⁺-GDP and Mg²⁺-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

Effect of local sugar and base geometry on <Superscript>13</Superscript>C and <Superscript>15</Superscript>N magnetic shielding anisotropy in DNA nucleosides

Density functional theory was employed to study the dependence of 13C and 15N magnetic shielding tensors on the glycosidic torsion angle (chi) and conformation of the sugar ring in 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine. In general, the magnetic shielding of the glycosidic nitrogens and the sugar carbons was found to depend on both the conformation of the sugar ring and chi. Our calculations indicate that the magnetic shielding anisotropy of the C6 atom in pyrimidine and the C8 atom in purine bases depends strongly on chi. The remaining base carbons were found to be insensitive to both sugar pucker and chi re-orientation. These results call into question the underlying assumptions of currently established methods for interpreting residual chemical shift anisotropies and 13C and 15N auto- and cross-correlated relaxation rates and highlight possible limitations of DNA applications of these methods.     

Backbone <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignment of full-length human uracil DNA glycosylase UNG2

Human uracil N-glycosylase isoform 2—UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1–92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93–313, 25.1 kDa). Here, we report the backbone ¹H, ¹³C, and ¹⁵N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.     

NMR <Superscript>1</Superscript>H,<Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and <Superscript>13</Superscript>C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here ¹H^{N, 15}N, and ¹³C resonance assignments for the 19.3 kDa (aa 1–169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS^G12C-GDP), using heteronuclear, multidimensional NMR spectroscopy. Backbone ¹H–¹⁵N correlations have been assigned for all non-proline residues, except for the first methionine residue.     

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C sequence specific backbone assignment of the vanadate inhibited hematopoietic tyrosine phosphatase

The sequence-specific backbone assignment of hematopoietic protein tyrosine phosphatase (HePTP; PTPN7) in presence of vanadate has been determined, based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. These assignments facilitate further studies of HePTP in the presence of inhibitors to target leukemia and provide further insights into the function of protein tyrosine phosphatases.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

Simultaneous CT-13C and VT-15N chemical shift labelling: Application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH

Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) ¹³C and variable time (VT) ¹⁵N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J_CC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2H_zN_z state, then prior to the VT chemical shift labelling period is converted into 2H_zN_y coherence. The VT -¹⁵N mode eliminates the effect of ¹ J _N,CO and ^1,2 J _N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH₂ groups, thus removing any potential overlap with the CH₃ signals. The CT-¹³CH₃,VT-¹⁵N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH₃NH and 3D ¹³C,¹⁵N HSQC-NOESY-CH₃NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH₃ and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH₃ and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.     

The influence of breeding colony and sex on mercury,selenium and lead levels and carbon and nitrogen stable isotope signatures in summer and winter feathers of <Emphasis Type="Italic">Calonectris</Emphasis> shearwaters

Contamination in marine foodwebs is nowadays of great environmental concern owing to the increasing levels of pollution in marine ecosystems from different anthropogenic sources. Seabirds can be used as indicators of regional contaminant patterns across large temporal and spatial scales. We analysed Hg, Se and Pb levels as well as stable isotope ratios of C (¹³C/¹²C, δ¹³C) and N (¹⁵N/¹⁴N, δ¹⁵N) in breeding- and winter-season feathers on males and females of two related shearwater species, providing information on spatiotemporal patterns of contaminants as well as the influence of the trophic ecology of these seabirds on contaminant levels. During the breeding season, Se and Pb concentrations were highest at the Cape Verde archipelago, showing no differences among the other colonies or between the sexes. However, Hg levels varied among colonies, being highest in the Mediterranean, probably resulting from the larger emissions and fallout of this pollutant in Europe. Feathers grown during breeding also showed sexual differences in Hg concentrations and δ¹³C. Differences in Hg concentration between sexes are mainly due to egg-laying decontamination in females. In contrast, differences in Hg among colonies are probably related to differences in trophic ecology, as indicated by δ¹³C and δ¹⁵N measurements. Contaminant concentrations in winter-grown feathers did not show any relationship with stable isotope values but were affected by contaminant loads associated with the breeding season. These findings suggest that the interpretation of contaminant levels of migratory species from feathers moulted out of the breeding season should be made with caution because those values could reflect exposures to contaminants acquired during the breeding season. We conclude that factors other than feeding ecology may play an important role in the interpretation of contaminant levels and their annual dynamics at several spatial scales. Consideration of the relevant temporal context provided by isotopic signatures and contaminant concentrations is important in deciphering contaminant information based on various tissues. Xavier Ruiz: deceased 27 April 2008.