<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.     

Empirical correlation between protein backbone <Superscript>15</Superscript>N and <Superscript>13</Superscript>C secondary chemical shifts and its application to nitrogen chemical shift re-referencing

The linear analysis of chemical shifts (LACS) has provided a robust method for identifying and correcting ¹³C chemical shift referencing problems in data from protein NMR spectroscopy. Unlike other approaches, LACS does not require prior knowledge of the three-dimensional structure or inference of the secondary structure of the protein. It also does not require extensive assignment of the NMR data. We report here a way of extending the LACS approach to ¹⁵N NMR data from proteins, so as to enable the detection and correction of inconsistencies in chemical shift referencing for this nucleus. The approach is based on our finding that the secondary ¹⁵N chemical shift of the backbone nitrogen atom of residue i is strongly correlated with the secondary chemical shift difference (experimental minus random coil) between the alpha and beta carbons of residue i − 1. Thus once alpha and beta ¹³C chemical shifts are available (their difference is referencing error-free), the ¹⁵N referencing can be validated, and an appropriate offset correction can be derived. This approach can be implemented prior to a structure determination and can be used to analyze potential referencing problems in database data not associated with three-dimensional structure. Application of the LACS algorithm to the current BMRB protein chemical shift database, revealed that nearly 35% of the BMRB entries have δ ¹⁵N values mis-referenced by over 0.7 ppm and over 25% of them have δ ¹H^N values mis-referenced by over 0.12 ppm. One implication of the findings reported here is that a backbone ¹⁵N chemical shift provides a better indicator of the conformation of the preceding residue than of the residue itself. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

Carbonyl ¹³C′ relaxation is dominated by the contribution from the ¹³C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the ¹⁵N relaxation data. Furthermore, the non-axial nature of the ¹³C′ CSA tensor results in a T₁/T₂ value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the ¹³C′ T₁/T₂ improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the ¹⁵N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Critical test of some computational methods for prediction of NMR <Superscript>1</Superscript>H and <Superscript>13</Superscript>C chemical shifts

Performance of 18 DFT functionals (B1B95, B3LYP, B3PW91, B97D, BHandHLYP, BMK, CAM-B3LYP, HSEh1PBE, M06-L, mPW1PW91, O3LYP, OLYP, OPBE, PBE1PBE, tHCTHhyb, TPSSh, wB97xD, VSXC) in combinations with six basis sets (cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, aug-cc-pVTZ, IGLO-II, and IGLO-III) and three methods for calculating magnetic shieldings (GIAO, CSGT, IGAIM) was tested for predicting ¹H and ¹³C chemical shifts for 25 organic compounds, for altogether 86 H and 88 C atoms. Proton shifts varied between 1.03 ppm to 12.00 ppm and carbon shifts between 7.87 ppm to 209.28 ppm. It was found that the best method for calculating ¹³C shifts is PBE1PBE/aug-cc-pVDZ with CSGT or IGAIM approaches (mae?=?1.66 ppm), for ¹H the best results were obtained with HSEh1PBE, mPW1PW91, PBE1PBE, CAM-B3LYP, and B3PW91 functionals with cc-pVTZ basis set and with CSGT or IGAIM approaches (mae?=?0.28 ppm). We found that often larger basis sets do not give better results for chemical shifts. The best basis sets for calculating ¹H and ¹³C chemical shifts were cc-pVTZ and aug-cc-pVDZ, respectively. CSGT and IGAIM NMR approaches can perform really well and are in most cases better than popular GIAO approach.

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Graphical Abstract Mean absolute errors for ¹H and ¹³C chemical shifts and computational times of neutral toluene molecule with aug-cc-pVDZ basis set and CSGT approach

     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of Saccharomyces cerevisiae type 1 thioredoxin in the oxidized state by solution NMR spectroscopy

Thioredoxins (Trx) are ubiquitous proteins that regulate several biochemical processes inside the cell. Trx is an important player, displaying oxidoreductase activity and helping to keep and regulate the oxidative state of the cellular environment. Trx also participates in the regulation of many cellular functions, such as DNA synthesis, protection against oxidative stress, cell cycle and signal transduction. The oxidized Trx is the target for another set of proteins, such as thioredoxin reductase (TrR), which used the reductive potential of NADPH. The oxidized state of Trx also plays important role in regulation of redox state in the cells. In this regard, the oxidized form of Trx is a putative conformer that contributes to the cellular redox environment. Here we report the chemical shift assignments (¹H, ¹³C and ¹⁵N) in solution at 15 °C. We also showed the secondary structure analysis of the oxidized form of yeast thioredoxin (yTrx1) as basis for future NMR studies of protein–target interactions and dynamics. The assignment was done at low concentration (200 µM) because it is important to keep intact the water cavity.     

<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

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     

<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.     

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.     

<Superscript>31</Superscript>P NMR correlation maps of <Superscript>18</Superscript>O/<Superscript>16</Superscript>O chemical shift isotopic effects for phosphometabolite labeling studies

Intramolecular correlations among the ¹⁸O-labels of metabolic oligophosphates, mapped by J-decoupled ³¹P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the ¹⁸O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of ¹⁸O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological usefulness of the J-decoupled ³¹P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally ¹⁸O labeled in perfused mammalian hearts.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C assignment of the amyloidogenic protein medin using fast-pulsing NMR techniques

Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576.     

Use of quantitative <Superscript>1</Superscript>H NMR chemical shift changes for ligand docking into barnase

¹H NMR complexation-induced changes in chemical shift (CIS) of HN protons have been used to characterize the complexes of barnase with the deoxyoligonucleotides d(GC) and d(CGAC). Quantitative shift changes are used not only to locate the most probable binding site (using ring-current shifts), but also to determine the orientation of the ligand within the binding site, based on a more complete shift calculation including bond magnetic anisotropies and electric field effects. For both ligands, the guanine is in the same binding site cleft, in the same position as identified in the crystal structure of the d(CGAC) complex. By contrast, a previous X-ray crystal structure of the d(GC) complex showed the ligand in the mouth of the active site, rather than at the guanyl-specific site, implying that the location may be an artifact of the crystallisation process.     

<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.     

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.     

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.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.