NMR assignment of the domain 513–651 from the SARS-CoV nonstructural protein nsp3

Sequence-specific NMR assignments of an internal domain of the protein nsp3, nsp3(513–651), which is a part of the SARS coronavirus (SARS-CoV) replicase polyprotein, have been determined, using triple-resonance NMR experiments with the uniformly [¹³C,¹⁵N]-labeled protein. The complete assignments (>99%) provide the basis for the ongoing three-dimensional structure determination.     

1H, 15N, and 13C NMR signal assignments of IIIGlc, a signal-transducing protein of Escherichia coli, using three-dimensional triple-resonance techniques

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) of Escherichia coli. Virtually complete (98%) backbone 1H, 15N, and 13C nuclear magnetic resonance (NMR) signal assignments were determined by using a battery of triple-resonance three-dimensional (3D) NMR pulse sequences. In addition, nearly complete (1H, 95%; 13C, 85%) side-chain 1H and 13C signal assignments were obtained from an analysis of 3D 13C HCCH-COSY and HCCH-TOCSY spectra. These experiments rely almost exclusively upon one- and two-bond J couplings to transfer magnetization and to correlate proton and heteronuclear NMR signals. Hence, essentially complete signal assignments of this 168-residue protein were made without any assumptions regarding secondary structure and without the aid of a crystal structure, which is not yet available. Moreover, only three samples, one uniformly 15N-enriched, one uniformly 15N/13C-enriched, and one containing a few types of amino acids labeled with 15N and/or 13C, were needed to make the assignments. The backbone assignments together with the 3D 15N NOESY-HMQC and 13C NOESY-HMQC data have provided extensive information about the secondary structure of this protein [Pelton, J.G., Torchia, D.A., Meadow, N.D., Wong, C.-Y., & Roseman, S (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3479-3488]. The nearly complete set of backbone and side-chain atom assignments reported herein provide a basis for studies of the three-dimensional structure and dynamics of IIIGlc as well as its interactions with a variety of membrane and cytoplasmic proteins.     

1H, 15N and 13C backbone and side chain chemical shifts of human ASC (apoptosis-associated speck-like protein containing a CARD domain)

The backbone and side chain resonance assignments of human ACS (∼22 KD), apoptosis-associated speck-like protein containing a caspase recruitment domain and a pyrin domain, have been determined by triple-resonance NMR techniques.     

Automated protein fold determination using a minimal NMR constraint strategy

Determination of precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium-accuracy fold information can often provide key clues about protein evolution and biochemical function(s). In this article we describe a largely automatic strategy for rapid determination of medium-accuracy protein backbone structures. Our strategy derives from ideas originally introduced by other groups for determining medium-accuracy NMR structures of large proteins using deuterated, (13)C-, (15)N-enriched protein samples with selective protonation of side-chain methyl groups ((13)CH(3)). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side-chain (15)N, H(N) resonances, and side-chain (13)CH(3) methyl resonances. These assignments are determined automatically by the program AutoAssign using backbone triple resonance NMR data, together with Spin System Type Assignment Constraints (STACs) derived from side-chain triple-resonance experiments. The program AutoStructure then derives conformational constraints using these chemical shifts, amide (1)H/(2)H exchange, nuclear Overhauser effect spectroscopy (NOESY), and residual dipolar coupling data. The total time required for collecting such NMR data can potentially be as short as a few days. Here we demonstrate an integrated set of NMR software which can process these NMR spectra, carry out resonance assignments, interpret NOESY data, and generate medium-accuracy structures within a few days. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A.     

ABACUS, a direct method for protein NMR structure computation via assembly of fragments

The ABACUS algorithm obtains the protein NMR structure from unassigned NOESY distance restraints. ABACUS works as an integrated approach that uses the complete set of available NMR experimental information in parallel and yields spin system typing, NOE spin pair identities, sequence specific resonance assignments, and protein structure, all at once. The protocol starts from unassigned molecular fragments (including single amino acid spin systems) derived from triple-resonance (1)H/(13)C/(15)N NMR experiments. Identifications of connected spin systems and NOEs precede the full sequence specific resonance assignments. The latter are obtained iteratively via Monte Carlo-Metropolis and/or probabilistic sequence selections, molecular dynamics structure computation and BACUS filtering (A. Grishaev and M. Llinás, J Biomol NMR 2004;28:1-10). ABACUS starts from scratch, without the requirement of an initial approximate structure, and improves iteratively the NOE identities in a self-consistent fashion. The procedure was run as a blind test on data recorded on mth1743, a 70-amino acid genomic protein from M. thermoautotrophicum. It converges to a structure in ca. 15 cycles of computation on a 3-GHz processor PC. The calculated structures are very similar to the ones obtained via conventional methods (1.22 A backbone RMSD). The success of ABACUS on mth1743 further validates BACUS as a NOESY identification protocol.     

NMR assignment of the nonstructural protein nsp3(1066–1181) from SARS-CoV

Sequence-specific NMR assignments of the globular core comprising the residues 1066–1181 within the non-structural protein nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [¹³C, ¹⁵N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure determination. A preliminary identification of regular secondary structures has been derived from the ¹³C chemical shifts.     

Peptides and the development of double- and triple-resonance solid-state NMR of aligned samples.

Peptides have been instrumental in the development of solid-state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid-state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple-resonance methods are described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples are presented.     

Backbone 1H, 15N, and 13C resonance assignments of the Helicobacter pylori acyl carrier protein

One of the small proteins from Helicobacter pylori, acyl carrier protein (ACP), was investigated by NMR. ACP is related to various cellular processes, especially with the biosynthesis of fatty acid. The basic NMR resonance assignment is a prerequisite for the validation of a heterologous protein interaction with ACP in H. pylori. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the H. pylori ACP are reported using double- and triple-resonance techniques. About 97% of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 76 of the 78 non-proline residues are clarified through sequential- and specific- assignments. In addition, four helical regions were clearly identified on the basis of the resonance assignments.     

Backbone and sidechain 1H, 15N and 13C assignments of Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa

The backbone and side chain resonance assignments of the Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa have been determined based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. This assignment is the first step towards the determination of the 3-dimensional structure of TpbA.     

Resonance assignment of As-p18, a fatty acid binding protein secreted by developing larvae of the parasitic nematode Ascaris suum

As-p18 is produced and secreted by larvae of the parasitic nematode Ascaris suum as they develop within their eggs. The protein is a member of the fatty acid binding protein (FABP) family found in a wide range of eukaryotes, but is distinctive in that it is secreted from the synthesizing cell and has predicted additional structural features not previously seen in other FABPs. As-p18 and similar proteins found only in nematodes have therefore been designated ‘nemFABPs’. Sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments were established for the 155 amino acid recombinant protein (18.3 kDa) in complex with oleic acid, using a series of three-dimensional triple-resonance heteronuclear NMR experiments. The secondary structure of As-p18 is predicted to be very similar to other FABPs, but the protein has extended loops that have not been observed in other FABPs whose structures have so far been solved.     

Backbone and sidechain 1H, 15N and 13C assignments of the NLRP7 pyrin domain

The resonance assignments of the human NLRP7 PYD domain have been determined based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. This assignment is the first step towards the 3D structure determination of the NLRP7 PYD domain.     

NMR assignments of the yellow fever virus envelope protein domain III

Nearly complete backbone and sidechain resonance assignments have been obtained for the third domain, residues S288–K398, of the envelope protein from the Asibi strain of yellow fever virus using double- and triple-resonance spectroscopy.     

NMR assignments of the sylvatic dengue 1 virus envelope protein domain III

Nearly complete backbone and side chain resonance assignments have been obtained for the third domain, residues M289–K400, of the envelope protein from the sylvatic strain (P72–1244) of the dengue 1 virus, containing mutations N336S and E370K, using double- and triple-resonance spectroscopy.     

Backbone and side chain 1H, 15N and 13C assignments of the KSR1 CA1 domain

The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [¹³C, ¹⁵N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain.     

Backbone 1H, 15N, and 13C resonance assignment of HP1242 from Helicobacter pylori

One of the small proteins from Helicobacter pylori, HP1242, was investigated by the solution nuclear magnetic resonance (NMR) spectroscopy. HP1242 is known as a 76-residue conserved hypothetical protein and its function cannot be identified based on sequence homology. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the HP1242 are reported using double- and triple-resonance techniques. About 95 % of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 75 non-Proline residues of the 76 residues are clarified through sequential- and specific- assignments. In addition, three helical regions were clearly identified on the basis of the resonance assignments.     

NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling

NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successful overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

An efficient high-throughput resonance assignment procedure for structural genomics and protein folding research by NMR.

Sequence specific resonance assignment is the primary requirement for all investigations of proteins by NMR methods. In the present postgenomic era where structural genomics and protein folding have occupied the center stage of NMR research, there is a high demand on the speed of resonance assignment, whereas the presently available methods based either on NOESY or on some triple-resonance experiments are rather slow. They also have limited success with unfolded proteins because of the lack of NOEs, and poor dispersion of amide and carbon chemical shifts. This paper describes an efficient approach to rapid resonance assignment that is suitable for both folded and unfolded proteins, making use of the triple-resonance experiments described recently [HNN and HN(C)N]. It has three underlying principles. First, the experiments exploit the (15)N chemical shift dispersions which are generally very good for both folded and unfolded proteins, along two of the three dimensions; second, they directly display sequential amide and (15)N correlations along the polypeptide chain, and third, the sign patterns of the diagonal and the sequential peaks originating from any residue are dependent on the nature of the adjacent residues, especially the glycines and the prolines. These lead to so-called "triplet fixed points" which serve as starting points and/or check points during the course of sequential walks, and explicit side chains assignment becomes less crucial for unambiguous backbone assignment. These features significantly enhance the speed of data analysis, reduce the amount of experimentation required, and thus result in a substantially faster and unambiguous assignment. Following the amide and (15)N assignments, the other proton and carbon assignments can be obtained in a straightforward manner, from the well-established three-dimensional triple-resonance experiments. We have successfully tested the new approach with different proteins in the molecular mass range of 10-22 kDa, and for illustration, we present here the backbone results on the HIV-1 protease-tethered dimer (molecular mass approximately 22 kDa), both in the folded and in the unfolded forms, the two ends of the folding funnel. We believe that the new assignment approach will be of great value for both structural genomics and protein folding research by NMR.     

1H, 15N and 13C assignments of the calcium bound S100P

To determine the three-dimensional solution structure of the calcium bound S100P protein, the backbone and side chain resonance assignments of the S100P protein have been reported based on triple-resonance experiments using uniformly [¹³C, ¹⁵N]-labeled calcium bound protein.     

Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein.

The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys-Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an alphahelix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain chi1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.