Density functional calculations of chemical shielding of backbone <Superscript>15</Superscript>N in helical residues of protein G

We performed density functional calculations of backbone ¹⁵N chemical shielding tensors in selected helical residues of protein G. Here we describe a computationally efficient methodology to include most of the important effects in the calculation of chemical shieldings of backbone ¹⁵N. We analyzed the role of long-range intra-protein electrostatic interactions by comparing models with different complexity in vacuum and in charge field. Our results show that the dipole moment of the α-helix can cause significant deshielding of ¹⁵N; therefore, it needs to be considered when calculating ¹⁵N chemical shielding. We found that it is important to include interactions with the side chains that are close in space when the charged form for ionizable side chains is adopted in the calculation. We also illustrate how the ionization state of these side chains can affect the chemical shielding tensor elements. Chemical shielding calculations using a 8-residue fragment model in vacuum and adopting the charged form of ionizable side chains yield a generally good agreement with experimental data.     

Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic 15N chemical shielding anisotropy tensors

Density functional theory was employed to study the influence of O-phosphorylation of serine, threonine, and tyrosine on the amidic ¹⁵N chemical shielding anisotropy (CSA) tensor in the context of the complex chemical environments of protein structures. Our results indicate that the amidic ¹⁵N CSA tensor has sensitive responses to the introduction of the phosphate group and the phosphorylation-promoted rearrangement of solvent molecules and hydrogen bonding networks in the vicinity of the phosphorylated site. Yet, the calculated ¹⁵N CSA tensors in phosphorylated model peptides were in range of values experimentally observed for non-phosphorylated proteins. The extent of the phosphorylation induced changes suggests that the amidic ¹⁵N CSA tensor in phosphorylated proteins could be reasonably well approximated with averaged CSA tensor values experimentally determined for non-phosphorylated amino acids in practical NMR applications, where chemical surrounding of the phosphorylated site is not known a priori in majority of cases. Our calculations provide estimates of relative errors to be associated with the averaged CSA tensor values in interpretations of NMR data from phosphorylated proteins.     

Influence of N-H...O and O-H...O hydrogen bonds on the (17)O, (15)N and (13)C chemical shielding tensors in crystalline acetaminophen: a density functional theory study

A computational investigation was carried out to characterize the (17)O, (15)N and (13)C chemical shielding tensors in crystalline acetaminophen. We found that N-H...O and O-H...O hydrogen bonds around the acetaminophen molecule in the crystal lattice have different influences on the calculated (17)O, (15)N and (13)C chemical shielding eigenvalues and their orientations in the molecular frame of axes. The calculations were performed with the B3LYP method and 6-311++G(d, p) and 6-311+G(d) standard basis sets using the Gaussian 98 suite of programs. Calculated chemical shielding tensors were used to evaluate the (17)O, (15)N, and (13)C NMR chemical shift tensors in crystalline acetaminophen, which are in reasonable agreement with available experimental data. The difference between the calculated NMR parameters of the monomer and molecular clusters shows how much hydrogen-bonding interactions affect the chemical shielding tensors of each nucleus. The computed (17)O chemical shielding tensor on O(1), which is involved in two intermolecular hydrogen bonds, shows remarkable sensitivity toward the choice of the cluster model, whereas the (17)O chemical shielding tensor on O(2) involved in one N-H...O hydrogen bond, shows smaller improvement toward the hydrogen-bonding interactions. Also, a reasonably good agreement between the experimentally obtained solid-state (15)N and (13)C NMR chemical shifts and B3LYP/6-311++G(d, p) calculations is achievable only in molecular cluster model where a complete hydrogen-bonding network is considered. Moreover, at the B3LYP/6-311++G(d, p) level of theory, the calculated (17)O, (15)N and (13)C chemical shielding tensor orientations are able to reproduce the experimental values to a reasonably good degree of accuracy.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

Calculation of chemical shift anisotropy in proteins

Individual peptide groups in proteins must exhibit some variation in the chemical shift anisotropy (CSA) of their constituent atoms, but not much is known about the extent or origins of this dispersion. Direct spectroscopic measurement of CSA remains technically challenging, and theoretical methods can help to overcome these limitations by estimating shielding tensors for arbitrary structures. Here we use an automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) approach to compute ¹⁵N, ¹³C′ and ¹H chemical shift tensors for human ubiquitin and the GB1 and GB3 fragments of staphylococcal protein G. The average and range of variation of the anisotropies is in good agreement with experimental estimates from solid-state NMR, and the variation among residues is somewhat smaller than that estimated from solution-state measurements. Hydrogen-bond effects account for much of the variation, both between helix and sheet regions, and within elements of secondary structure, but other effects (including variations in torsion angles) may play a role as well.     

Density functional calculations of 15N chemical shifts in solvated dipeptides

We performed density functional calculations to examine the effects of solvation, hydrogen bonding, backbone conformation, and the side chain on 15N chemical shielding in proteins. We used N-methylacetamide (NMA) and N-formyl-alanyl-X (with X being one of the 19 naturally occurring amino acids excluding proline) as model systems. In addition, calculations were performed for selected fragments from protein GB3. The conducting polarizable continuum model was employed to include the effect of solvent in the density functional calculations. Our calculations for NMA show that the augmentation of the polarizable continuum model with the explicit water molecules in the first solvation shell has a significant influence on isotropic 15N chemical shift but not as much on the chemical shift anisotropy. The difference in the isotropic chemical shift between the standard beta-sheet and alpha-helical conformations ranges from 0.8 to 6.2 ppm depending on the residue type, with the mean of 2.7 ppm. This is in good agreement with the experimental chemical shifts averaged over a database of 36 proteins containing >6100 amino acid residues. The orientation of the 15N chemical shielding tensor as well as its anisotropy and asymmetry are also in the range of values experimentally observed for peptides and proteins.     

Vibrational averaging of chemical shift anisotropies in model peptides

The effects of chemical shift anisotropy (CSA) are evident in line-shapes or side-band analysis in solid-state NMR, in the observed line positions in partially oriented samples, and in relaxation effects in liquid-state studies. In all of these cases, the effective shielding tensor is influenced by fast vibrational averaging in addition to larger-amplitude internal motions and to overall libration or rotation. Here we compute the contributions of vibrational averaging (including zero-point motions) to the CSA relaxation strengths for the nitrogen and carbonyl carbon in two simple peptide models, and for snapshots taken from a path-integral simulation of a small protein. Because the ¹⁵N shielding tensor is determined by all the atoms of the peptide group, it is less influenced by vibrational motion than (for example) the N–H dipolar interaction, which is more sensitive to the motion of the light hydrogen atom. Computed order parameters for CSA averaging are hence much closer to unity than are N–H dipolar order parameters. This leads to a reduction by about 9% in the magnitude of the amide nitrogen CSA that is needed to fit liquid-state relaxation data. Similar considerations apply to the carbonyl carbon shielding tensor, but in this case the differences between dipolar and CSA averaging are smaller. These considerations will be important for making comparisons between CSA tensors extracted from various NMR experiments, and for comparisons to quantum chemical calculations carried out on static conformers.     

Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection

15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor.     

Backbone dynamics of green fluorescent protein and the effect of histidine 148 substitution

Green fluorescent protein (GFP) and its mutants have become valuable tools in molecular biology. GFP has been regarded as a very stable and rigid protein with the beta-barrel shielding the chromophore from the solvent. Here, we report the 15N nuclear magnetic resonance (NMR) studies on the green fluorescent protein (GFPuv) and its mutant His148Gly. 15N NMR relaxation studies of GFPuv show that most of the beta-barrel of GFP is rigid on the picosecond to nanosecond time scale. For several regions, including the first alpha-helix and beta-sheets 3, 7, 8, and 10, increased hydrogen-deuterium exchange rates suggest a substantial conformational flexibility on the microsecond to millisecond time scales. Mutation of residue 148 located in beta-sheet 7 is known to have a strong impact on the fluorescence properties of GFPs. UV absorption and fluorescence spectra in combination with 1H-15N NMR spectra indicate that the His148Gly mutation not only reduces the absorption of the anionic chromophore state but also affects the conformational stability, leading to the appearance of doubled backbone amide resonances for a number of residues. This suggests the presence of two conformations in slow exchange on the NMR time scale in this mutant.     

Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of <Superscript>15</Superscript>N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR

Magic-angle spinning solid-state NMR measurements of ¹⁵N longitudinal paramagnetic relaxation enhancements (PREs) in ¹³C,¹⁵N-labeled proteins modified with Cu²⁺-chelating tags can yield multiple long-range electron-nucleus distance restraints up to ~20 Å (Nadaud et al. in J Am Chem Soc 131:8108–8120, 2009). Using the EDTA-Cu²⁺ K28C mutant of B1 immunoglobulin binding domain of protein G (GB1) as a model, we investigate the effects on such measurements of intermolecular electron-nucleus couplings and intrinsic metal binding sites, both of which may potentially complicate the interpretation of PRE data in terms of the intramolecular protein fold. To quantitatively assess the influence of intermolecular ¹⁵N-Cu²⁺ interactions we have determined a nearly complete set of longitudinal ¹⁵N PREs for a series of microcrystalline samples containing ~10, 15 and 25 mol percent of the ¹³C,¹⁵N-labeled EDTA-Cu²⁺-tagged protein diluted in a matrix of diamagnetic natural abundance GB1. The residual intermolecular interactions were found to be minor on the whole and account for only a fraction of the relatively small but systematic deviations observed between the experimental ¹⁵N PREs and corresponding values calculated using protein structural models for residues furthest removed from the EDTA-Cu²⁺ tag. This suggests that these deviations are also caused in part by other factors not related to the protein structure, such as the presence in the protein of intrinsic secondary sites capable of binding Cu²⁺ ions. To probe this issue we performed a Cu²⁺ titration study for K28C-EDTA GB1 monitored by 2D ¹⁵N-¹H solution-state NMR, which revealed that while for Cu²⁺:protein molar ratios of ≤ 1.0 Cu²⁺ binds primarily to the high-affinity EDTA tag, as anticipated, at even slightly super-stoichiometric ratios the Cu²⁺ ions can also associate with side-chains of aspartate and glutamate residues. This in turn is expected to lead to enhanced PREs for residues located in the vicinity of the secondary Cu²⁺ binding sites, and indeed many of these residues were ones found to display the elevated longitudinal ¹⁵N PREs in the solid phase.     

Speeding up direct <Superscript>15</Superscript>N detection: hCaN 2D NMR experiment

Experiments detecting low gyromagnetic nuclei have recently been proposed to utilize the relatively slow relaxation properties of these nuclei in comparison to ¹H. Here we present a new type of ¹⁵N direct-detection experiment. Like the previously proposed CaN experiment (Takeuchi et al. in J Biomol NMR 47:271–282, 2010), the hCaN experiment described here sequentially connects amide ¹⁵N resonances, but utilizes the initial high polarization and the faster recovery of the ¹H nucleus to shorten the recycling delay. This allows recording 2D ¹⁵N-detected NMR experiments on proteins within a few hours, while still obtaining superior resolution for ¹³C and ¹⁵N, establishing sequential assignments through prolines, and at conditions where amide protons exchange rapidly. The experiments are demonstrated on various biomolecules, including the small globular protein GB1, the 22 kDa HEAT2 domain of eIF4G, and an unstructured polypeptide fragment of NFAT1, which contains many SerPro sequence repeats.     

Solution structure of the conserved hypothetical protein Rv2302 from Mycobacterium tuberculosis

The Mycobacterium tuberculosis protein Rv2302 (80 residues; molecular mass of 8.6 kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparallel beta-sheet core with one C-terminal alpha-helix (A61 to A75) nestled against its side. Hydrophobic interactions between residues in the alpha-helix and beta-strands 3 and 4 hold the alpha-helix near the beta-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {(1)H}-(15)N heteronuclear nuclear Overhauser effects indicate that the protein's core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G13 to H19 in the (1)H-(15)N heteronuclear single quantum correlation spectrum suggests that this region, a loop between beta-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.     

Probing 13C chemical shielding tensors in cryptolepine and two bromo-substituted analogs for antiplasmodial activity

Density functional theory calculations were applied to investigate ¹³C chemical shielding tensors in cryptolepine and its bromo-substituted analogs, 2-bromocryptolepine and 2,7-dibromocryptolepine. The fact that bromo-substituted cryptolepine shows higher antiplasmodial activity than cryptolepine raises the question of whether this effect can be related to the electronic properties around carbon atoms. The results show that changes to the principal components of the shielding tensors upon substitution are significant. In particular, σ ₃₃ is the most affected tensor for carbons in the substituted ring, which could be related to the increased antiplasmodial activity of bromosubstituted cryptolepine. The analyses were also focused on atomic charges and dipole moment.     

Determination of the spatial structure of insectotoxin 15A from Buthus erpeus by (1)H-NMR spectroscopy data]

The solution structure of insectotoxin 15A (35 residues) from scorpion Buthus eupeus was determined on the basis of 386 interproton distance restraints 12 hydrogen-bonding restraints and 113 dihedral angle restraints derived from 1H NMR experiments. A group of 20 structures was calculated with the distance geometry program DIANA followed by the restrained energy minimization with the program CHARMM. The atomic RMS distribution about the mean coordinate position is 0.64 +/- 0.11 A for the backbone atoms and 1.35 +/- 0.20 A for all atoms. The structure contains an alpha-helix (residues 10-20) and a three-stranded antiparallel beta-sheet (residues 2-5, 24-28 and 29-33). A pairing of the eight cysteine residues of insectotoxin 15A was established basing on NMR data. Three disulfide bridges (residues 2-19, 16-31 and 20-33) connect the alpha-helix with the beta-sheet, and the fourth one (5-26) joins beta-strands together. The spatial fold of secondary structure elements (the alpha-helix and the beta-sheet) of the insectotoxin 15A is very similar to those of the other short and long scorpion toxins in spite of a low (about 20%) sequence homology.     

MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21

We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2T_N polarization transfer delay for labeling of ¹⁵N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in ¹⁵N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)_NHs, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161–166, 2005). Therefore, cross peak overlap is diminished while the time period during which the ¹⁵N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19–0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.     

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.     

Theoretical investigation of hydrogen bonding effects on oxygen, nitrogen, and hydrogen chemical shielding and electric field gradient tensors of chitosan/HI salt

A density functional theory study has been carried out to calculate the (17)O, (15)N, (13)C, and (1)H chemical shielding as well as (17)O, (14)N, and (2)H electric field gradient tensors of chitosan/HI type I salt. These calculations were performed using the B3LYP functional and 6-311++G (d,p) and 6-31++G (d,p) basis sets. Calculated EFG and chemical shielding tensors were used to evaluate the (17)O, (14)N, and (2)H nuclear quadruple resonance, NQR, and (17)O, (15)N, (13)C, and (1)H nuclear magnetic resonance, NMR, parameters in the cluster model, which are in good agreement with the available experimental data. The difference in the isotropic shielding (sigma(iso)) and quadrupole coupling constant (C(Q)) between monomer and target molecule in the cluster was analyzed in detail. It was shown that both EFG and CS tensors are sensitive to hydrogen-bonding interactions, and calculating both tensors is an advantage. A different influence of various hydrogen bond types, N-Hcdots, three dots, centeredI, O-Hcdots, three dots, centeredI, and N-Hcdots, three dots, centeredO was observed on the calculated CS and EFG tensors. On the basis of this study, nitrogen and O-6 are the most important nuclei to confirm crystalline structure of chitosan/HI. These nuclei have large change in their CS and EFG tensors because of forming intermolecular hydrogen bonds. Moreover, the quantum chemical calculations indicated that the intermolecular hydrogen-bonding interactions play an essential role in determining the relative orientation of CS and EFG tensors of O-6 and nitrogen atoms in the molecular frame axes.     

Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments

Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low γ nuclei, such as ¹³C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel ¹⁵N direct-detection experiments. The CAN experiment sequentially connects amide ¹⁵N resonances using ¹³C^α chemical shift matching, and the CON experiment connects the preceding ¹³C′ nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding ¹³C-detecting experiment, NCA-DIPAP or NCO-IPAP (Bermel et al. 2006b; Takeuchi et al. 2008). However, the disadvantage due to the lower γ is counteracted by the slower ¹⁵N transverse relaxation during detection, the possibility for more efficient decoupling in both dimensions, and relaxation optimized properties of the pulse sequences. As a result, the median S/N in the ¹⁵N observe CAN experiment is 16% higher than in the ¹³C observe NCA-DIPAP experiment. In addition, significantly higher sensitivity was observed for those residues that are hard to detect in the NCA-DIPAP experiment, such as Gly, Ser and residues with high-field C^α resonances. Both CAN and CON experiments are able to detect Pro resonances that would not be observed in conventional proton-detected experiments. In addition, those experiments are free from problems of incomplete deuterium-to-proton back exchange in amide positions of perdeuterated proteins expressed in D₂O. Thus, these features and the superior resolution of ¹⁵N-detected experiments provide an attractive alternative for main chain assignments. The experiments are demonstrated with the small model protein GB1 at conditions simulating a 150 kDa protein, and the 52 kDa glutathione S-transferase dimer, GST.     

Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta-sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha-helix in the N-state. There is evidence for some fixed side-chain--side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.     

Solution structure and interactions of the Escherichia coli cell division activator protein CedA

CedA is a protein that is postulated to be involved in the regulation of cell division in Escherichia coli and related organisms; however, little biological data about its possible mode of action are available. Here we present a three-dimensional structure of this protein as determined by NMR spectroscopy. The protein is made up of four antiparallel beta-strands, an alpha-helix, and a large unstructured stretch of residues at the N-terminus. It shows structural similarity to a family of DNA-binding proteins which interact with dsDNA via a three-stranded beta-sheet, suggesting that CedA may be a DNA-binding protein. The putative binding surface of CedA is predominantly positively charged with a number of basic residues surrounding a groove largely dominated by aromatic residues. NMR chemical shift perturbations and gel-shift experiments performed with CedA confirm that the protein binds dsDNA, and its interaction is mediated primarily via the beta-sheet.