1H, 13C, and 15N resonance assignment of the cAMP-regulated phosphoprotein endosulfine-alpha in free and micelle-bound states

¹³C, ¹⁵N, and ¹H chemical shift assignments are presented for the cAMP-regulated phosphoprotein endosulfine-alpha in its free and micelle-bound states. Secondary chemical shift analysis demonstrates formation of four helices in the micelle-bound state, which are not present in the absence of detergent.     

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     

1H, 13C and 15N backbone and side chain resonance assignments of human interleukin 1α

As part of our NMR structure determination of the human Interleukin-1α, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C and ¹⁵N nuclei.     

1H, 15N, and 13C NMR chemical shift assignments for the Ig3 domain of palladin

As part of our NMR structure determination of the palladin Ig3 domain, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C, and ¹⁵N nuclei.     

Triple Resonance MAS NMR with (13C, 15N) Labelled Molecules: Reduced Dimensionality Data Acquisition Via 13C-15N Heteronuclear Two-Spin Coherence Transfer Pathways

A reduced dimensionality magic angle spinning solid-state NMR experimental protocol for obtaining chemical shift correlation spectra of dipolar coupled nuclei in uniformly (¹³C, ¹⁵N) labelled biological systems is described and demonstrated. The method involves a mapping of the evolution frequencies of heteronuclear ¹³C-¹⁵N zero- and double-quantum coherences. In comparison to a reduced dimensionality procedure involving the simultaneous incrementation of two single-quantum chemical shift evolution periods, the approach described here could be potentially advantageous for minimising the heat dissipated in the probe by high power ¹H decoupling in experiments requiring long t ₁ acquisition times.     

1H, 13C, and 15N backbone and side-chain chemical shift assignments for the 29 kDa human galectin-1 protein dimer

Galectin-1 is an important regulator of leukocyte function and tumor angiogenesis. Recently, this lectin has been identified as a molecular target for the potent angiogenesis inhibitor anginex. Here, we report ¹H, ¹³C, and ¹⁵N chemical shift assignments for human galectin-1 as determined by using heteronuclear triple resonance NMR spectroscopy.     

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.     

1H, 13C and 15N resonance assignments and peptide binding site chemical shift perturbation mapping for the Escherichia coli redox enzyme chaperone DmsD

Herein are reported the mainchain ¹H, ¹³C and ¹⁵N chemical shift assignments and amide ¹⁵N relaxation data for Escherichia coli DmsD, a 23.3 kDa protein responsible for the correct folding and translocation of the dimethyl sulfoxide reductase enzyme complex. In addition, the observed amide chemical shift perturbations resulting from complex formation with the reductase subunit DmsA leader peptide support a model in which the 44 residue peptide makes extensive contacts across the surface of the DmsD protein.     

Synthetic analogues of the histidine–chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex

Mg(II)–porphyrin–ligand and (bacterio)chlorophyl–ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. ¹H, ¹³C and ¹⁵N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9–5.2 ppm ¹H, 0.2–5.5 ppm ¹³C and 2.1–27.2 ppm ¹⁵N coordination shifts were measured for selectively labeled [1,3-¹⁵N]-Im, [1,3-¹⁵N,2-¹³C]-Im and [1,3-¹⁵N,1,2-¹³C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the ¹H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a–histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.Abbreviations (B)Chl a (bacterio)chlorophyll a - HMBC heteronuclear multiple bond correlation - Im imidazole - LH light-harvesting - 1-MeIm 1-methylimidazole - Mg(II)-Por Mg(II)-porphyrin macrocycle - OEP 2,3,7,8,12,13,17,18-octaethylporphyrin     

Sequence specific 1H, 13C and 15N resonance assignments of hahellin in 8 M urea

The sequence specific ¹H, ¹³C and ¹⁵N resonance assignments of hahellin in 8 M urea-denatured state have been accomplished by NMR spectroscopy. Secondary chemical shift analysis reveals the native-like propensities for β-rich conformation in the denatured state.     

Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving ¹³C and ¹⁵N spins have been developed for this purpose, ¹H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular ¹H-¹³C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved ¹H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR, simplifies the ¹H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced ¹H chemical shift resolution allows the assignment of overlapping H and H resonances in Ser, identifies the ¹H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed.     

NMR assignment of the N-terminal region of human La free and in complex with RNA

¹H, ¹⁵N and ¹³C chemical shift assignments are presented for the N-terminal region of human La protein, in the apo and 5′-UUUU RNA-bound state. Secondary structure analysis shows conformational changes in the interdomain linker upon complex formation.     

Two-and three-dimensional HCN experiments for correlating base and sugar resonances in 15N, 13C-labeled RNA oligonucleotides

Summary New 2D and 3D ¹H-¹³C-¹⁵N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in ¹³C-and ¹⁵N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective ¹³C-refocusing and ¹⁵N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D ¹H-¹⁵N and 3D¹H-¹³C-¹⁵N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra-and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.     

RefDB: a database of uniformly referenced protein chemical shifts

RefDB is a secondary database of reference-corrected protein chemical shifts derived from the BioMagResBank (BMRB). The database was assembled by using a recently developed program (SHIFTX) to predict protein ¹H, ¹³C and ¹⁵N chemical shifts from X-ray or NMR coordinate data of previously assigned proteins. The predicted shifts were then compared with the corresponding observed shifts and a variety of statistical evaluations performed. In this way, potential mis-assignments, typographical errors and chemical referencing errors could be identified and, in many cases, corrected. This approach allows for an unbiased, instrument-independent solution to the problem of retrospectively re-referencing published protein chemical shifts. Results from this study indicate that nearly 25% of BMRB entries with ¹³C protein assignments and 27% of BMRB entries with ¹⁵N protein assignments required significant chemical shift reference readjustments. Additionally, nearly 40% of protein entries deposited in the BioMagResBank appear to have at least one assignment error. From this study it evident that protein NMR spectroscopists are increasingly adhering to recommended IUPAC ¹³C and ¹⁵N chemical shift referencing conventions, however, approximately 20% of newly deposited protein entries in the BMRB are still being incorrectly referenced. This is cause for some concern. However, the utilization of RefDB and its companion programs may help mitigate this ongoing problem. RefDB is updated weekly and the database, along with its associated software, is freely available at http://redpoll.pharmacy.ualberta.ca and the BMRB website.     

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC??C and 3D C??NCA with sequential ¹³C acquisitions, 3D NHH and 3D NC??H with sequential ¹H acquisitions and 3D CANH and 3D C??NH with broadband ¹³C?C¹⁵N mixing are demonstrated using microcrystalline samples of the ??1 immunoglobulin binding domain of protein G (GB1) and the chicken ??-spectrin SH3 domain.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

Secondary structure and 1H, 13C and 15N resonance assignments of the Escherichia coli YaeT POTRA domain

We report the first ¹H, ¹³C and ¹⁵N chemical shift assignments and secondary structure of the Escherichia coli YaeT POTRA domain; a domain found in the Omp85 family of proteins which is critical for insertion and folding of outer membrane proteins in Gram-negative bacteria.     

NMR assignment of the C-terminal actin-binding domain of talin

Talin is a large dimeric 270 kDa adapter protein which binds the cytoplasmic face of a subset of integrin β-subunits and couples them to the actin cytoskeleton. Here we report the near complete ¹⁵N, ¹³C and ¹H chemical shift assignments for the C-terminal actin-binding domain.