Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic ¹H ²/¹³C ² or ¹H ¹/¹⁵N ¹ with aliphatic ¹³C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [¹⁵N, ¹H]- and/or [¹³C, ¹H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [¹⁵N, ¹H]-TROSY-H(N)C^ar experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.     

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].     

SARS Coronavirus Unique Domain: Three-Domain Molecular Architecture in Solution and RNA Binding

Nonstructural protein 3 of the severe acute respiratory syndrome (SARS) coronavirus includes a “SARS-unique domain” (SUD) consisting of three globular domains separated by short linker peptide segments. This work reports NMR structure determinations of the C-terminal domain (SUD-C) and a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution; in SUD-NM, there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel-shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observations of ¹⁵N-labeled proteins further resulted in delineation of RNA binding sites (i.e., in SUD-M, a positively charged surface area with a pronounced cavity, and in SUD-C, several residues of an anti-parallel β-sheet). Overall, the present data provide evidence for molecular mechanisms involving the concerted actions of SUD-M and SUD-C, which result in specific RNA binding that might be unique to the SUD and, thus, to the SARS coronavirus.     

NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23–230)

A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly ¹⁵N, ¹³C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable ¹⁵N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse ¹⁵N relaxation rates of unfolded polypeptides in high resolution constant-time [¹H, ¹⁵N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the ¹⁵N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence.     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

Multinuclear NMR resonance assignments and the secondary structure of Escherichia coli thioesterase/protease I: A member of a new subclass of lipolytic enzymes

Escherichia coli thioesterase/protease I is a 183 amino acid protein with a molecular mass of 20500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a new GDSLS consensus motif, of which no structure has yet been determined. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytically active. From analysis of a set of heteronuclear multidimensional NMR spectra with uniform and specific amino acid labeled protein samples, we have obtained near-complete resonance assignments of the backbone ¹H,¹³ C and ¹⁵N nuclei (BMRB databank accession number 4060). The secondary structure of E. coli thioesterase/protease I was further deduced from the consensus chemical shift indices, backbone short- and medium-range NOEs, and amide proton exchange rates. The protein was found to consist of four -strands and seven -helices, arranged in alternate order. The four -strands were shown to form a parallel -sheet. The topological arrangement of the -strands of -1x, +2x, +1x appears to resemble that of the core region of the hydrolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of -strands and the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioesterase/protease I at present.     

HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins

A TROSY-based triple-resonance pulse scheme is described which correlates backbone ¹H and ¹⁵N chemical shifts of an amino acid residue with the ¹⁵N chemical shifts of both the sequentially preceding and following residues. The sequence employs ¹ J _NC and ² J _NC couplings in two sequential magnetization transfer steps in an `out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly ²H/¹³C/¹⁵N-labelled proteins in the 30-kDa range.     

(H)NCAHA and (H)CANNH experiments for the determination of vicinal coupling constants related to the ϕ-torsion angle

Summary A set of three-dimensional triple-resonance experiments is described which provide , , and coupling constants. The pulse sequences generate E.COSY-like multiplet patterns and comprise a magnetization transfer from the amide proton to the α-proton or vice versa via the directly bound heteronuclei. For residues with the ¹H^α spin resonating close to the H₂O signal, a modified HNCA experiment can be employed to measure the vicinal ¹H^N,¹H^α couplings. Ambiguities associated with the conversion of values into ϕ-angle constraints for protein structure determination can be resolved with the knowledge of the heteronuclear ³J-couplings. In favourable cases, stereospecific assignments of glycine α-protons can be obtained by employing the experiments described here in combination with NOE data. The methods are applied to flavodoxin from Desulfovibrio vulgaris.     

Improved resolution in triple-resonance spectra by nonlinear sampling in the constant-time domain

Summary Nonlinear sampling along the constant-time dimension is applied to the constant-time HNCO spectrum of the dimerization domain of Ga14. Nonlinear sampling was used for the nitrogen dimension, while the carbon and proton dimensions were sampled linearly. A conventional ct-HNCO spectrum is compared with a nonlinearly sampled spectrum, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension. Nonlinearly sampled data are processed by maximum entropy reconstruction. It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time. The constant intensity of the signal in the constant-time dimension allows for a variety of sampling schedules. A schedule of randomly distributed sampling points yields the best results. This general method can be used to significantly increase the quality of heteronuclear constant-time spectra.