Complete Sequence-specific Assignments Research Articles

Backbone and side chain NMR assignments of the H-NOX domain from Nostoc sp. in complex with BAY58-2667 (cinaciguat).

Soluble guanylate cyclase (sGC) enzyme is activated by the gaseous signaling agent nitric oxide (NO) and triggers the conversion of GTP (guanosine 5'-triphosphate) to cGMP (cyclicguanylyl monophosphate). It contains the heme binding H-NOX (heme-nitric oxide/oxygen binding) domain which serves as the sensor of NO and it is highly conserved across eukaryotes and bacteria as well. Many research studies focus on the synthesis of chemical compounds bearing possible therapeutic action, which mimic the heme moiety and activate the sGC enzyme. In this study, we report a preliminary solution NMR (Nuclear Magnetic Resonance) study of the H-NOX domain from Nostoc sp. cyanobacterium in complex with the chemical sGC activator cinaciguat (BAY58-2667). An almost complete sequence-specific assignment of its 1H, 15N and 13C resonances was obtained and its secondary structure predicted by TALOS+.

Sequence specific 1H, 13C and 15N resonance assignments of the C-terminal domain of human γS-crystallin.

The high solubility and stability of crystallins present in the human eye lens maintains its transparency and refractive index with negligible protein turnover. Monomeric γ-crystallins and oligomeric β-crystallins are made up of highly homologous double Greek key domains. These domains are symmetric and possess higher stability as a result of the complex topology of individual Greek key motifs. γS-crystallin is one of the most abundant structural βγ-crystallins present in the human eye lens. In order to understand the structural stability of individual domains of human γS-crystallin in isolation vis-à-vis full length protein, we set out to structurally characterize its C-terminal domain (abbreviated hereafter as γS-CTD) by solution NMR. In this direction, we have cloned, over-expressed, isolated and purified the γS-CTD. The 2D [15N-1H] HSQC recorded with uniformly 13C/15N labeled γS-CTD showed a highly dispersed spectrum indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-CTD using a suite of heteronuclear 3D NMR experiments.

Sequence-Specific Solution NMR Assignments of the β-Barrel Insertase BamA to Monitor Its Conformational Ensemble at the Atomic Level.

β-barrel outer membrane proteins (Omps) are key functional components of the outer membranes of Gram-negative bacteria, mitochondria, and plastids. In bacteria, their biogenesis requires the β-barrel-assembly machinery (Bam) with the central insertase BamA, but the exact translocation and insertion mechanism remains elusive. The BamA insertase features a loosely closed gating region between the first and last β-strand 16. Here, we describe ∼70% complete sequence-specific NMR resonance assignments of the transmembrane region of the BamA β-barrel in detergent micelles. On the basis of the assignments, NMR spectra show that the BamA barrel populates a conformational ensemble in slow exchange equilibrium, both in detergent micelles and lipid bilayer nanodiscs. Individual conformers can be selected from the ensemble by the introduction of a C-terminal strand extension, single-point mutations, or specific disulfide cross-linkings, and these modifications at the barrel seam are found to be allosterically coupled to sites at the entire barrel circumference. The resonance assignment provides a platform for mechanistic studies of BamA at atomic resolution, as well as for investigating interactions with potential antibiotic drugs and partner proteins.

Sequence specific 1H, 13C and 15N resonance assignments of a cataract-related variant G57W of human γS-crystallin.

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [15N-1H]-HSQC spectrum recorded with uniformly 13C/15N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.

NMR study of non-structural proteins--part II: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV).

Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/β-fold.

NMR study of non-structural proteins--part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV).

Macro domains are ADP-ribose-binding modules present in all eukaryotic organisms, bacteria and archaea. They are also found in non-structural proteins of several positive strand RNA viruses such as alphaviruses. Here, we report the high yield expression and preliminary structural analysis through solution NMR spectroscopy of the macro domain from New World Mayaro Alphavirus. The recombinant protein was well-folded and in a monomeric state. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure determined by TALOS+.

Sequence-specific 1H-NMR Assignment and Determination of the Secondary Structure of Hainantoxin-III from the Spider Ornithoctonus hainana

Hainantoxin-III (HNTX-III) purified from the venom of the spider Ornithoctonus hainana is a novel neurotoxin preferentially inhibiting tetrodotoxin-sensitive voltage-gated sodium channels in rat dorsal root ganglion cells. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequencespecific assignments of proton resonances in the 1H-NMR spectra were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O or D2O. All the backbone protons and more than 95% of the side-chain protons have been assigned by dαN, dβN, and dNN connectivities in NOESY spectrum. Furthermore, the secondary structure of HNTX-III was identified from NMR data. It consists mainly of a short triple-stranded antiparallel β-sheet formed by Asp7 to Cys9, Tyr21 to Ser23, and Lys27 to Val30. Because HNTX-III shares high sequence identity (>70%) with HWTX-I and HNTX-I, we proposed that they all share a structural scaffold known as the inhibitor cystine knot architectural motif. This study provides a basis for the further determination of the solution conformation of HNTX-III.

1H, 15N, 13C assignment and secondary structure determination of two domains of La protein from D. discoideum

Biosynthesis of RNA polymerase III transcripts requires binding of the La protein at their 3' end. La is an abundant nuclear RNA-binding protein which protects the nascent transcripts from 3' exonuclease degradation. Here, we report the high yield expression and preliminary structural analysis through NMR spectroscopy of two recombinant RNA binding domains (La motif and NRRM) from the La protein of Dictyostelium discoideum. Both recombinant protein constructs were well-folded and allowed for an almost complete sequence-specific assignment of the (15)N and (13)C labeled domains and their secondary structure prediction using PECAN online tool.

Selective lighting up of segments around Gly, Ala and Ser/Thr in proteins

Direct detection of (13) C nucleus can be used as a valuable alternative where (1) H detection poses a challenge due to relaxation effects, chemical exchange and poor chemical shift dispersion. In this context, we have designed a suite of 2D (13) C(α) -detected hNCA experiments that provide sequential correlations of (13) C(α) with (15) N on one hand and efficient spectroscopic labeling of certain groups of residues, namely, Gly, Ala, Ser and Thr, on the other. These residues act as checkpoints in the sequential walk, which in turn offer new possibilities of backbone assignment of small proteins from a set of 2D experiments, thereby providing great economy in terms of spectrometer time. The direct identification of peptide segments around Gly, Ala, Ser and Thr residues along a protein chain will be highly valuable for deriving important information on sites of ligand binding, phosphorylation, inhibitor/substrate binding, understanding protein folding pathways, comprehending local conformational dynamics etc. without having to obtain complete sequence-specific assignments, which can be time consuming and at times formidable, especially in large proteins. We have illustratively demonstrated the multifaceted applications of these variants of 2D experiments on ubiquitin and M-crystallin. We foresee that these 2D hNCA experiments will provide economic and efficient strategies for studying the structure and function of proteins.

Facile backbone (1H, 15N, 13Ca, and 13C′) assignment of 13C/15N-labeled proteins using orthogonal projection planes of HNN and HN(C)N experiments and its automation

Recently, we introduced an efficient high-throughput protocol for backbone assignment of small folded proteins based on two-dimensional (2D) projections of HN(C)N suite of experiments and its automation [Borkar et al., J. Biomol. NMR 2011, 50(3), 285-297]. This strategy provides complete sequence-specific assignment of backbone ((1)H, (15)N, (13)C(α), and (13)C') resonances in less than a day; thus, it has great implications for high-throughput structural proteomics. However, in cases when such small folded protein exhibits substantial amide (1)H shift degeneracy (typically seen in alpha-helical proteins), the strategy may fail or lead to ambiguities. Another limitation is with respect to the identification of checkpoints from the variants of 2D-hncNH spectrum. For example, a protein with many GG, GA, AA, SS, TS, TT, and TS types of dipeptide stretches along its sequence, thus the identification of NH cross-peak corresponding to second G, A, S, or T becomes difficult. In this backdrop, we present here two improvements to enhance the utility of the proposed high-throughput AUTOmatic Backbone Assignment protocol: (i) use of 2D-hNnH spectrum and its variants that display additional (1)H-(15)N correlations and thus help to resolve ambiguities arising because of amide (1)H shift degeneracy and (ii) optimization of the τ(CN) delay in the 2D-hncNH experiment that, when properly adjusted, is observed to help remove ambiguities in the identification of the checkpoints. These improvements have also been incorporated in the automation program AUTOmatic Backbone Assignment. Finally, the performance of the strategy and the automation has been demonstrated using the chicken SH3 domain protein.

Study of angiotensin-(1–7) vasoactive peptide and its β-cyclodextrin inclusion complexes: Complete sequence-specific NMR assignments and structural studies

We report the complete sequence-specific hydrogen NMR assignments of vasoactive peptide angiotensin-(1–7) (Ang-(1–7)). Assignments of the majority of the resonances were accomplished by COSY, TOCSY, and ROESY peak coordinates at 400 MHz and 600 MHz. Long-side-chain amino acid spin system identification was facilitated by long-range coherence transfer experiments (TOCSY). Problems with overlapped resonance signals were solved by analysis of heteronuclear 2D experiments (HSQC and HMBC). Nuclear Overhauser effects (NOE) results were used to probe peptide conformation. We show that the inclusion of the angiotensin-(1–7) tyrosine residue is favored in inclusion complexes with β-cyclodextrin. QM/MM simulations at the DFTB/UFF level confirm the experimental NMR findings and provide detailed structural information on these compounds in aqueous solution.

Nuclear Magnetic Resonance Studies on Huwentoxin-XI from the Chinese Bird Spider <italic>Ornithoctonus huwena:</italic><sup>15</sup>N Labeling and Sequence-specific <sup>1</sup>H, <sup>15</sup>N Nuclear Magnetic Resonance Assignments

Huwentoxin-XI purified from the Chinese bird spider Ornithoctonus huwena is a toxin with both antiprotease activity and potassium channel blocking activity. To determine its solution structure, huwentoxin-XI was expressed in a yeast eukaryotic expression system and studied by NMR. The 15N labeling strategy was used to facilitate the process of resonance assignments. The nearly complete sequence-specific assignments of proton and nitrogen resonances were obtained by analyzing a series of two-dimensional (2D) and three-dimensional (3D) spectra, including DQF-COSY, TOCSY, NOESY, 15N-1H HSQC, 15N-1H HNHA, 15N-1H HNHB, 15N-1H TOCSY-HSQC and 15N-1H NOESY-HSQC spectra. Secondary structure analysis of huwentoxin-XI showed that it mainly contains an N-terminal 310-helix from Thr3 to Arg5 and a C-terminal alpha-helix from Gln45 to Cys52, plus a triple-stranded antiparallel beta-sheet of Glu18-Asn23, Thr26-Ile31 and Asn40-Lys41. These studies provide a solid basis for the final structure determination of huwentoxin-XI.

Automated Protein NMR Structure Determination in Crude Cell-Extract

A fully automated, NOE-based NMR structure determination of a uniformly 13C,15N-labeled protein was achieved in crude cell-extract, without purification of the overexpressed protein. Essentially complete sequence-specific assignments were obtained using triple resonance experiments, based on the high intensity of the resonances from the overexpressed protein relative to those of the background. For the collection of NOE distance constraints, efficient discrimination between NOE cross peaks from the target protein and background signals was achieved using the programs ATNOS and CANDID. In the iterative ATNOS/CANDID procedure, the identification of the desired protein NOEs is initially guided by the self-consistency of the protein NOE-network. Although the intensities of the signals in this network vary over a wide range, and are in many instances comparable to or smaller than those of the background, the first cycle of calculations resulted in the correct global polypeptide fold, and the structure was then refined in six subsequent cycles using the intermediate NMR structures for additional guidance. The experience gained with this work demonstrates that the ATNOS/CANDID procedure for automatic protein structure determination is highly robust and reliable in the presence of intense background signals, and might thus also represent a platform for future protein structure determinations in physiological fluids.

Sequence-specific Assignment of <sup>1</sup>H-NMR Resonance and Determination of the Secondary Structure of Jingzhaotoxin-I

Jingzhaotoxin-I (JZTX-I) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by d alphaN, d alphadelta, d betaN and d NN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel beta-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I (HWTX-I) and hainantoxin-IV (HNTX-IV), whose structures in solution have previously been reported.

NMR Structure of the Integral Membrane Protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(δ 1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel β-barrel, with successive pairs of β-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded β-sheet beyond the height of the barrel. This protruding β-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.

How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.

Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA β-barrel from those of OmpX. Overall, the present data demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.

Sequence‐specific 13C NMR assignments in a neurotoxin (candoxin) from Bungarus candidus

AbstractWe report 13C NMR assignments of a weak neurotoxin called candoxin, using heteronuclear single quantum correlation (HSQC) experiments with their natural abundance in the protein. Almost complete sequence‐specific 13C NMR assignments were achieved. Chemical shift indices of 13Cα, 13Cβ and 1Hα were used to characterize the secondary structural segments in the protein. A consensus plot of chemical shift indices revealed five β‐strands as the major structural elements in the toxin. Copyright © 2001 John Wiley & Sons, Ltd.

Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles.

The (2)H,(13)C,(15)N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30 degrees C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The (13)C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.