<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignments for pyrazinoic acid binding domain of ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Ribosomal protein S1 of Mycobacterium tuberculosis (MtRpsA) binds to ribosome and mRNA, and plays significant role in the regulation of translation initiation, conventional protein synthesis and transfer-messenger RNA (tmRNA) mediated trans-translation. It has been identified as the target of pyrazinoic acid (POA), a bactericidal moiety from hydrolysis of pyrazinamide, which is a mainstay of combination therapy for tuberculosis. POA prevented the interactions between the C-terminal S1 domain of MtRpsA (residues 280–368, MtRpsA^CTD_S1) and tmRNA; so that POA can inhibit the trans-translation, which is a key component of multiple quality control pathways in bacteria. However, the details of molecular mechanism and dynamic characteristics for MtRpsA^CTD_S1 interactions with POA, tmRNA or mRNA are still unclear. Here we present the ¹H, ¹⁵N, ¹³C resonance assignments of MtRpsA^CTD_S1 as well as the secondary structure information based on backbone chemical shifts, which lay foundation for further solution structure determination, dynamic properties characterization and interactions investigation between MtRpsA^CTD_S1 and tmRNA, RNA or POA.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C backbone resonance assignment of the C-terminal domain of enzyme I from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone ¹H, ¹⁵N and ¹³C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the ¹H–¹⁵N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments of the C-terminal domain of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> TolA protein

Vibrio cholerae is the bacterial causative agent of the human disease cholera. Non-pathogenic bacterium can be converted to pathogenic following infection by a filamentous phage, CTXΦ, that carries the cholera toxin encoding genes. A crucial step during phage infection requires a direct interaction between the CTXΦ minor coat protein (pIII^CTX) and the C-terminal domain of V. cholerae TolA protein (TolAIIIvc). In order to get a better understanding of TolA function during the infection process, we have initiated a study of the V. cholerae TolAIII domain by 2D and 3D heteronuclear NMR. With the exception of the His-tag (H123–H128), 97 % of backbone ¹H, ¹⁵N and ¹³C resonances were assigned and the side chain assignments for 92 % of the protein were obtained (BMRB deposit with accession number 25689).     

(3, 2)D <Superscript>1</Superscript>H, <Superscript>13</Superscript>C BIRD<Superscript>r,X</Superscript>-HSQC-TOCSY for NMR structure elucidation of mixtures: application to complex carbohydrates

Overlap of NMR signals is the major cause of difficulties associated with NMR structure elucidation of molecules contained in complex mixtures. A 2D homonuclear correlation spectroscopy in particular suffers from low dispersion of ¹H chemical shifts; larger dispersion of ¹³C chemical shifts is often used to reduce this overlap, while still providing the proton–proton correlation information e.g. in the form of a 2D ¹H, ¹³C HSQC-TOCSY experiment. For this methodology to work, ¹³C chemical shift must be resolved. In case of ¹³C chemical shifts overlap, ¹H chemical shifts can be used to achieve the desired resolution. The proposed (3, 2)D ¹H, ¹³C BIRD^r,X-HSQC-TOCSY experiment achieves this while preserving singlet character of cross peaks in the F₁ dimension. The required high-resolution in the ¹³C dimension is thus retained, while the cross peak overlap occurring in a regular HSQC-TOCSY experiment is eliminated. The method is illustrated on the analysis of a complex carbohydrate mixture obtained by depolymerisation of a fucosylated chondroitin sulfate isolated from the body wall of the sea cucumber Holothuria forskali.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of an unusual Ca<Superscript>2+</Superscript>-binding protein from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> in its apo form

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of an unusual Ca²⁺-binding protein from Entamoeba histolytica (EhCaBP6) in its apo form as a prelude to its structural and functional characterization.     

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>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments and secondary structure analysis of translation initiation factor 1 from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of infection in humans. P. aeruginosa can acquire resistance against multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones, and multidrug resistance is increasing in this organism which makes treatment of the infections difficult and expensive. This has led to the unmet need for discovery of new compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Translation initiation factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis, and its structure is unknown. Here we report the ¹H, ¹³C and ¹⁵N chemical shift assignments of Pa-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified five β-strands with an unusually extended β-strand at the C-terminal end of the protein and one short α-helix arranged in the sequential order β1–β2–β3–α1–β4–β5. This is further supported by ¹⁵N–{¹H} hetero NOEs. These secondary structure elements suggest the Pa-IF1 adopts the typical β-barrel structure and is composed of an oligomer-binding motif.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of a calcium-binding protein from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

Near-complete backbone resonance assignments of acid-denatured human cytochrome <Emphasis Type="Italic">c</Emphasis> in dimethylsulfoxide: a prelude to studying interactions with phospholipids

Human cytochrome c plays a central role in the mitochondrial electron transfer chain and in the intrinsic apoptosis pathway. Through the interaction with the phospholipid cardiolipin, cytochrome c triggers release of pro-apoptotic factors, including itself, from the mitochondrion into the cytosol of cells undergoing apoptosis. The cytochrome c/cardiolipin complex has been extensively studied through various spectroscopies, most recently with high-field solution and solid-state NMR spectroscopies, but there is no agreement between the various studies on key structural features of cytochrome c in its complex with cardiolipin. In the present study, we report backbone ¹H, ¹³C, ¹⁵N resonance assignments of acid-denatured human cytochrome c in the aprotic solvent dimethylsulfoxide. These have led to the assignment of a reference 2D ¹H-¹⁵N HSQC spectrum in which out of the 99 non-proline residues 87% of the backbone amides are assigned. These assignments are being used in an interrupted H/D exchange strategy to map the binding site of cardiolipin on human cytochrome c.     

Partial solid-state NMR <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of a perdeuterated back-exchanged seven-transmembrane helical protein <Emphasis Type="Italic">Anabaena</Emphasis> Sensory Rhodopsin

Anabaena Sensory Rhodopsin (ASR) is a unique photochromic membrane-embedded photosensor which interacts with soluble transducer and is likely involved in a light-dependent gene regulation in the cyanobacterium Anabaena sp. PCC 7120. We report partial spectroscopic ¹H, ¹³C and ¹⁵N assignments of perdeuterated and back-exchanged ASR reconstituted in lipids. The reported assignments are in general agreement with previously determined assignments of carbon and nitrogen resonances in fully protonated samples. Because the back-exchange was performed on ASR in a detergent-solubilized state, the location of detected residues reports on the solvent accessibility of ASR in detergent. A comparison with the results of previously published hydrogen/exchange data collected on the ASR reconstituted in lipids, suggests that the protein has larger solvent accessible surface in the detergent-solubilized state.     

Anionic polymers of the cell wall of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> subsp. <Emphasis Type="Italic">subtilis</Emphasis> VKM B-501<Superscript>T</Superscript>

Teichoic acid and disaccharide-1-phosphate polymer were identified in the cell walls of Bacillus subtilis subsp. subtilis VKM B-501^T. The teichoic acid represents 1,3-poly(glycerol phosphate) 80% substituted by α-D-glucopyranose residues at O-2 of glycerol. The linear repeating unit of disaccharide-1-phosphate polymer contains the residues of β-D-glucopyranose, N-acetyl-α-D-galactosamine, and phosphate and has the following structure: -6)-β-D-Glcp-(1→3)-α-D-GalpNAc-(1-P-. The structures of two anionic polymers were determined by chemical and NMR-spectroscopic methods. The ¹H- and ¹³C-NMR spectral data on disaccharide-1-phosphate polymer are presented for the first time.     

Natural Abundance Fourier Transform of <Superscript>13</Superscript>C Nuclear Magnetic Resonance Spectra of Lysozyme

NUCLEAR magnetic resonance (NMR) studies of biopolymers have so far been limited to proton resonances. On the other hand, ¹³C NMR analysis is potentially a more powerful technique because of the larger range of chemical shifts (300 p.p.m. compared with 10 p.p.m. for ^XH NMR) and because the chemical shift of ¹³C NMR is more sensitive to the nature of the chemical bonds. The chief limitations of ¹³C NMR are low natural abundance (1·1%), low relative sensitivity (1·59% of proton) and long relaxation times (?1 min). These can be largely overcome, however, by the combined techniques of nuclear Overhauser enhancement and fourier transform spectroscopy. Thus, Gibbons et al.¹ obtained a simple and elegant spectral analysis of ‘Gramicidin S-A’, a decapeptide. We report here the first natural abundance ¹³C NMR spectrum of an enzyme. Lysozyme was chosen because its primary structure is known^2,3 and because its ¹H NMR spectrum has already been described⁴.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments of light organ-associated fatty acid-binding protein of Taiwanese fireflies

Fatty acid-binding proteins (FABPs) are a family of proteins that modulate the transfer of various fatty acids in the cytosol and constitute a significant portion in many energy-consuming cells. The ligand binding properties and specific functions of a particular type of FABP seem to be diverse and depend on the respective binding cavity as well as the cell type from which this protein is derived. Previously, a novel FABP (lcFABP; lc: Luciola cerata) was identified in the light organ of Taiwanese fireflies. The lcFABP was proved to possess fatty acids binding capabilities, especially for fatty acids of length C₁₄–C₁₈. However, the structural details are unknown, and the structure–function relationship has remained to be further investigated. In this study, we finished the ¹H, ¹⁵N and ¹³C chemical shift assignments of ¹⁵N/¹³C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, C_α, H_α, C and side chain C_β assignments. These results can provide the basis for further structural exploration of lcFABP.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments for a chemokine receptor-binding domain of FROUNT,a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain ¹H, ¹³C, and ¹⁵N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the ¹H–¹⁵N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.     

<Superscript>1</Superscript>H NMR analysis of <Emphasis Type="Italic">Citrus macrophylla</Emphasis> subjected to Asian citrus psyllid (<Emphasis Type="Italic">Diaphorina citri</Emphasis> Kuwayama) feeding

The Asian citrus psyllid (ACP) is a phloem-feeding insect that can host and transmit the bacterium Candidatus Liberibacter asiaticus (CLas), which is the putative causative agent of the economically important citrus disease, Huanglongbing (HLB). ACP are widespread in Florida, and are spreading in California; they are the primary mode of CLas transmission in citrus groves. To understand the effects of ACP feeding, different numbers of ACP [0 ACP (control), 5 ACP (low), 15–20 ACP (medium), and 25–30 ACP (high)] were allowed to feed on Citrus macrophylla greenhouse plants. After 7 days of feeding, leaves were collected and analyzed using ¹H NMR. Metabolite concentrations from leaves of trees with ACP feeding had higher variability than control trees. Many metabolites were higher in concentration in the low ACP feeding group relative to control; however, leaves from trees with high ACP feeding had lower concentrations of many metabolites relative to control, including many amino acids such as phenylalanine, arginine, isoleucine, valine, threonine, and leucine. These results suggest ACP density-dependent changes in primary metabolism that can be measured by ¹H NMR. The implications in plant defense are discussed.