<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.     

Crystal structures exploring the origins of the broader specificity of escherichia coli heat-labile enterotoxin compared to cholera toxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally and functionally related and share the same primary receptor, the GM1 ganglioside. Despite their extensive similarities, these two toxins exhibit distinct ligand specificities, with LT being more promiscuous than CT. Here, we have attempted to rationalize the broader binding specificity of LT and the subtle differences between the binding characteristics of LTs from human and porcine origins (mediated by their B subunit pentamers, hLTB and pLTB, respectively). The analysis is based on two crystal structures of pLTB in complexes with the pentasaccharide of its primary ligand, GM1, and with neolactotetraose, the carbohydrate determinant of a typical secondary ligand of LTs, respectively. Important molecular determinants underlying the different binding specificities of LTB and CTB are found to be contributed by Ser95, Tyr18 and Thr4 (or Ser4 of hLTB), which together prestabilize the binding site by positioning Lys91, Glu51 and the adjacent loop region (50-61) containing Ile58 for ligand binding. Glu7 and Ala1 may also play an important role. Many of these residues are closely connected with a recently identified second binding site, and there appears to be cross-talk between the two sites. Binding to N-acetyllactosamine-terminated receptors is further augmented by Arg13 (present in pLT and some hLT variants), as previously predicted.     

<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak

Ahnak is a ~?700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca²⁺ signaling in cardiomyocytes by interacting with the β subunit of the L-type Ca²⁺ channel (Ca_V1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete ¹H, ¹³C and ¹⁵N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the Ca_V1.2-Ahnak complex.     

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.     

Selective backbone labeling of proteins using {1,2-<Superscript>13</Superscript>C<Subscript>2</Subscript>}-pyruvate as carbon source

A simple isotope labeling approach for selective ¹³C/¹⁵N backbone labeling of proteins is described. Using {1,2-¹³C₂}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of ¹³C^α-¹³CO spin-pairs into the backbones of protein molecules with medium-to-high levels of ¹³C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a 7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein NMR applications are expected to benefit from the {1,2-¹³C₂}-pyruvate based protein production.     

(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>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.     

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     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

Pressure dependence of side chain <Superscript>13</Superscript>C chemical shifts in model peptides Ac-Gly-Gly-Xxx-Ala-NH<Subscript>2</Subscript>

For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of ¹³C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH₂ (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B ₁ and B ₂ is dependent on the type of atom and amino acid studied. For H^N, N and C^α the first order pressure coefficient B ₁ is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically ¹³C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

Carbonyl ¹³C′ relaxation is dominated by the contribution from the ¹³C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the ¹⁵N relaxation data. Furthermore, the non-axial nature of the ¹³C′ CSA tensor results in a T₁/T₂ value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the ¹³C′ T₁/T₂ improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the ¹⁵N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pressure-dependent <Superscript>13</Superscript>C chemical shifts in proteins: origins and applications

Pressure-dependent ¹³C chemical shifts have been measured for aliphatic carbons in barnase and Protein G. Up to 200 MPa (2 kbar), most shift changes are linear, demonstrating pressure-independent compressibilities. CH₃, CH₂ and CH carbon shifts change on average by +0.23, −0.09 and −0.18 ppm, respectively, due to a combination of bond shortening and changes in bond angles, the latter matching one explanation for the γ-gauche effect. In addition, there is a residue-specific component, arising from both local compression and conformational change. To assess the relative magnitudes of these effects, residue-specific shift changes for protein G were converted into structural restraints and used to calculate the change in structure with pressure, using a genetic algorithm to convert shift changes into dihedral angle restraints. The results demonstrate that residual ¹³Cα shifts are dominated by dihedral angle changes and can be used to calculate structural change, whereas ¹³Cβ shifts retain significant dependence on local compression, making them less useful as structural restraints.     

<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.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

<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.