Protein hydration studied with homonuclear 3D1H NMR experiments

Summary Homonuclear 3D¹H NOESY-TOCSY and 3D¹H ROESY-TOCSY experiments were used to resolve and assign nuclear Overhauser effect (NOE) cross peaks between the water signal and individual polypeptide proton resonances in H₂O solutions of the basic pancreatic trypsin inhibitor. Combined with a novel, robust water-suppression technique, positive and negative intermolecular NOEs were detected at 4°C. The observation of positive NOEs between water protons and protein protons enables more precise estimates of the very short residence times of the water molecules in the hydration sites on the protein surface.     

Improving solvent suppression in jump-return NOESY experiments

Summary The problems associated with solvent suppression in jump-return NOESY spectra and in particular the difficulties experienced with using short mixing times are examined. It is shown that the degree of water suppression depends critically on the extent of radiation damping of the water magnetisation during the mixing time of the NOESY sequence. A new jump-return NOESY sequence is proposed which incorporates field gradients and which achieves good levels of water suppression for all values of the mixing time, and for all increments of the NOESY experiment.     

A computationally efficient method for evaluating the gradient of 2D NOESY intensities

Summary A computationally efficient method for calculating the derivative of NOE intensities with respect to any parameter is presented. This method is based on an integral expression representing the gradient. We will derive this expression from first principles using standard perturbation expansion techniques, and show it to be equivalent to an analytical expression [Yip, P. and Case, D.A. (1989) J. Magn. Reson., 83, 643] Implementation of this method in a refinement scheme (NOE-MD) is also briefly mentioned.     

Binomial frequency response to non-binomial pulse sequences for efficient water suppression

Summary This article reports on the use of short-hard pulse and spin-lock pulse combinations giving a binomial-like frequency response for the measurement of NMR spectra in aqueous solutions of quite dilute samples. The pulse sequence proposed provides excellent water suppression and does not introduce any linear or higher order phase errors. Application to the measurement of 2D NOESY data of a 0.25 mM solution of a double-stranded DNA fragment is presented.     

Separation of intramolecular NOE and exchange peaks in water exchange spectroscopy using spin-echo filters

Summary A technique for separating intramolecular NOE and solvent-proton exchange peaks in exchange spectroscopy is demonstrated. This method utilizes the large differences in relaxation and coupling properties of water and macromolecules to separate the two effects. The spin-echo filter consists of a water-frequency selective 90° pulse followed by a spin-echo sequence. If the echo time is sufficiently long, protein resonances (e.g. CH protons) excited by the selective pulse are removed due to their much shorter T₂ values and J-coupling evolution. By combining the filter with exchange spectroscopy (EXSY) or water exchange (WEX) filter experiments, exchange peaks can be selectively observed. In this paper the filter is combined with a modified version of the WEX filter (WEX II filter) with 1D and 2D detection and applied to a zinc finger peptide and to staphylococcal nuclease, allowing estimation of the contribution of intramolecular NOEs to the exchange spectra.To whom correspondence should be addressed.     

Automated assignment and 3D structure calculations using combinations of 2D homonuclear and 3D heteronuclear NOESY spectra

The NOAH/DIAMOD suite uses feedback filtering and self-correcting distance geometry to generate 3D structures from unassigned NOESY spectra. In this study we determined the minimum set of experiments needed to generate a high quality structure bundle. Different combinations of 3D ¹⁵N-edited, ¹³C-edited HSQC-NOESY and 2D homonuclear ¹H-¹H NOESY spectra of the 77 amino acid protein, myeloid progenitor inhibitory factor-1 (MPIF-1) were used as input for NOAH/DIAMOD calculations. The quality of the assignments of NOESY cross peaks and the accuracy of the automatically generated 3D structures were compared to those obtained with a conventional manual procedure. Combining data from two types of experiments synergistically increased the number of peaks assigned unambiguously in both individual spectra. As a general trend for the accuracy of the structures we observed structural variations in the backbone fold of the final structures of about 2 Å for single spectral data, of 1 Å to 1.5 Å for double spectral data, and of 0.6 Å for triple spectral data sets. The quality of the assignments and 3D structures from the optimal data using all three spectra were similar to those obtained from traditional assignment methods with structural variations within the bundle of 0.6 Å and 1.3 Å for backbone and heavy atoms, respectively. Almost all constraints (97%) of the automatic NOESY cross peak assignments were cross compatible with the structures from the conventional manual assignment procedure, and an even larger proportion (99%) of the manually derived constraints were compatible with the automatically determined 3D structures. The two mean structures determined by both methods differed only by 1.3 Å rmsd for the backbone atoms in the well-defined regions of the protein. Thus NOAD/DIAMOD analysis of spectra from labeled proteins provides a reliable method for high throughput analysis of genomic targets.     

Full NMR assignment and revised structure for the capsular polysaccharide from Streptococcus pneumoniae type 15B

The capsular polysaccharide from Streptococcus pneumoniae Type 15B is a component of the 23-valent polysaccharide vaccine against pneumococcal disease. We report full NMR assignments for the native and de-O-acetylated polysaccharide, and confirm that the phosphorylated substituent is glycerol-2-phosphate rather than phosphocholine, located on O-3 of the side chain beta-Galp residue. The polysaccharide is O-acetylated on the terminal alpha-Gal residue, distributed between O-2, O-3, O-4 and O-6 in a ratio of 6:12:12:55, with approximately 15% of the repeat units not O-acetylated.     

Folding Topology of a Bimolecular DNA Quadruplex Containing a Stable Mini-hairpin Motif within the Diagonal Loop

We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G₃ACGTAGTG₃)₂ containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G₃T₄G₃)₂ dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T₄-loop sequence. A truncated version of the bimolecular quadruplex d(G₂ACGTAGTG₂)₂, with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.     

Statistical analysis of double NOE transfer pathways in proteins as measured in 3D NOE-NOE spectroscopy

Summary The recent development of three-dimensional NMR spectroscopy has alleviated the problem of overlap of resonances. However, also for the 3D experiments resonance assignment strategies have usually relied upon knowledge about spin systems, combined with information about short (sequential) distances. For doubly (¹⁵N/¹³C)-labelled molecules, a novel assignment strategy has been developed. In this paper we address the possibilities of an assignment strategy for proteins, based solely upon the use of NOE data. For this, the 3D NOE-NOE experiment seems most suitable. Therefore, we have made a theoretical evaluation of double NOE transfer pathways in 28 protein crystal structures. We identify 95 connectivities which are most likely to be observed as cross peaks in a 3D NOE-NOE spectrum of a protein. Given the occurrence of one of these 95 connectivities, we evaluate the chances of occurrence for the others. Analysis of these conditional probabilities allowed the construction of five patterns of related, highly correlated cross peaks which resemble the conventional idea of spin systems to some extent and may provide a basis for assignment and secondary structure analysis from 3D NOE-NOE data alone.Dedicated to the memory of Professor V.F. Bystrov     

Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12)

Tyrosine sulfation of the chemokine receptor CXCR4 enhances its interaction with the chemokine SDF-1alpha. Given similar post-translational modification of other receptors, including CCR5, CX3CR1 and CCR2b, tyrosine sulfation may be of universal importance in chemokine signaling. N-terminal domains from seven transmembrane chemokine receptors have been employed for structural studies of chemokine-receptor interactions, but never in the context of proper post-translational modifications known to affect function. A CXCR4 peptide modified at position 21 by expressed tyrosylprotein sulfotransferase-1 and unmodified peptide are both disordered in solution, but bind SDF-1alpha with low micromolar affinities. NMR and fluorescence polarization measurements showed that the CXCR4 peptide stabilizes dimeric SDF-1alpha, and that sulfotyrosine 21 binds a specific site on the chemokine that includes arginine 47. We conclude that the SDF-1alpha dimer preferentially interacts with receptor peptide, and residues beyond the extreme N-terminal region of CXCR4, including sulfotyrosine 21, make specific contacts with the chemokine ligand.     

State of water in starch-water systems in the gelatinization temperature range as investigated using dielectric relaxation spectroscopy

The dielectric response of native wheat starch-water slurries containing 5-60% starch (w/w) was measured in the frequency range of 0.2-20 GHz after heating the slurries to 7 different temperatures between 25 and 90 °C for 30 min. Three relaxations, with relaxation time range of 4-9 ps, 20-25 ps and 230-620 ps at 25 °C, were identified from the dielectric spectra of starch slurries. The fastest relaxation process (4-9 ps) was attributed to bulk water while the two slower relaxations were attributed to the confined water molecules present in the starch-water system. The amount of water exhibiting the slowest relaxation (230-620 ps) was calculated to be 0.08-0.16 g water/g starch, which was close to the monolayer water associated with wheat starch. Mobility of bulk water was significantly reduced (P < 0.001) upon gelatinization at low starch concentration (10% starch), but remained unaffected at higher starch concentrations. The mobility of two slower relaxing water species was not significantly influenced (P > 0.19) by gelatinization at all starch concentrations.     

A variable target intensity-restrained global optimization (VARTIGO) procedure for determining three-dimensional structures of polypeptides from NOESY data: Application to gramicidin-S

Summary A global optimization method for intensity-restrained structure refinement, based on variable target function (VTF) analysis, is illustrated using experimental data on a model peptide, gramicidin-S (GS) dissolved in DMSO. The method (referred to as VARTIGO for variable target intensity-restrained global optimization) involves minimization of a target function in which the range of NOE contacts is gradually increased in successive cycles of optimization in dihedral angle space. Several different starting conformations (including all-trans) have been tested to establish the validity of the method. Not all optimizations were successful, but these were readily identifiable from their large NOE R-factors. We also show that it is possible to simultaneously optimize the rotational correlation time along with the dihedral angles. The structural features of GS thus obtained from the successful optimizations are in excellent agreement with the available experimental data. A comparison is made with structures generated from an intensity-restrained single target function (STF) analysis. The results on GS suggest that VARTIGO refinement is capable of yielding better quality structures. Our work also underscores the need for a simultaneous analysis of different NOE R-factors in judging the quality of optimized structures. The NOESY data on GS in DMSO appear to provide evidence for the presence of two orientations for the ornithine side chain, in fast exchange. The NOESY spectra for this case were analyzed using a relaxation rate matrix which is a weighted average of the relaxation rate matrices for the individual conformations.     

NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain

Efficient formation of specific intermolecular interactions is essential for self-assembly of biological structures. The foldon domain is an evolutionarily optimized trimerization module required for assembly of the large, trimeric structural protein fibritin from phage T4. Monomers consisting of the 27 amino acids comprising a single foldon domain subunit spontaneously form a natively folded trimer. During assembly of the foldon domain, a monomeric intermediate is formed on the submillisecond time scale, which provides the basis for two consecutive very fast association reactions. Mutation of an intermolecular salt bridge leads to a monomeric protein that resembles the kinetic intermediate in its spectroscopic properties. NMR spectroscopy revealed essentially native topology of the monomeric intermediate with defined hydrogen bonds and side-chain interactions but largely reduced stability compared to the native trimer. This structural preorganization leads to an asymmetric charge distribution on the surface that can direct rapid subunit recognition. The low stability of the intermediate allows a large free-energy gain upon trimerization, which serves as driving force for rapid assembly. These results indicate different free-energy landscapes for folding of small oligomeric proteins compared to monomeric proteins, which typically avoid the transient population of intermediates.     

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2ⁿ×2ⁿ pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.     

Electrostatic contributions to the stability of the GCN4 leucine zipper structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK_a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK_a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK_a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK_a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK_a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK_a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.     

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Covariance NMR in higher dimensions: application to 4D NOESY spectroscopy of proteins

Elucidation of high-resolution protein structures by NMR spectroscopy requires a large number of distance constraints that are derived from nuclear Overhauser effects between protons (NOEs). Due to the high level of spectral overlap encountered in 2D NMR spectra of proteins, the measurement of high quality distance constraints requires higher dimensional NMR experiments. Although four-dimensional Fourier transform (FT) NMR experiments can provide the necessary kind of spectral information, the associated measurement times are often prohibitively long. Covariance NMR spectroscopy yields 2D spectra that exhibit along the indirect frequency dimension the same high resolution as along the direct dimension using minimal measurement time. The generalization of covariance NMR to 4D NMR spectroscopy presented here exploits the inherent symmetry of certain 4D NMR experiments and utilizes the trace metric between donor planes for the construction of a high-resolution spectral covariance matrix. The approach is demonstrated for a 4D (13)C-edited NOESY experiment of ubiquitin. The 4D covariance spectrum narrows the line-widths of peaks strongly broadened in the FT spectrum due to the necessarily short number of increments collected, and it resolves otherwise overlapped cross peaks allowing for an increase in the number of NOE assignments to be made from a given dataset. At the same time there is no significant decrease in the positive predictive value of observing a peak as compared to the corresponding 4D Fourier transform spectrum. These properties make the 4D covariance method a potentially valuable tool for the structure determination of larger proteins and for high-throughput applications in structural biology.     

Effects of ultraviolet radiation and water motion on the reef coral, Porites compressa Dana: a transplantation experiment

Scleractinian corals have adapted to live in habitats were the level of ultraviolet radiation (UVR, 280–400 nm) is extremely high. The putative photoprotective molecules called mycosporine-like amino acids (MAAs) contained in the corals' tissues absorb UVR and release it harmlessly as heat. MAA concentration in corals is quite plastic and correlates well with UVR dose, but other ecological factors such as water motion may influence MAA production as well. In this study, the effects of ambient UVR and water motion on MAA concentration and several physiological parameters of the reef coral Porites compressa Dana were investigated in a two by two factorial transplantation experiment. Replicate branches from nine morphologically distinct colonies were transplanted from the windward side of Coconut Island (Kaneohe Bay, HI) to a control area on the windward side (ambient water motion) and to an area on the leeward side (low water motion). The transplanted corals were placed under UV-opaque (UVO) or UV-transparent (UVT) filters fixed to the reef. Initially and at 3 and 6 weeks, coral branches were weighed to determine calcification rate and tissues were extracted in methanol for photosynthetic pigment and MAA analysis via high performance liquid chromatography (HPLC). UVR was a significant factor determining MAA concentration. When UVR was screened from the corals' environment, total MAA concentration decreased by 33% over 6 weeks. However, UVR-exposed corals moved to low water motion also decreased MAA levels, while UVR-exposed corals moved to the control area retained initial levels. Photosynthetic pigments and calcification rate were also significantly reduced in corals moved to low water motion. There was no UVR effect on photosynthetic pigments or calcification rate. This study provides evidence that water motion is important for the maintenance of MAAs. However, there were interesting colony-specific patterns in MAA composition and response to the UVR treatment; some colonies had high total concentrations of MAAs in all treatments, while others displayed a pronounced UVR effect. Also, each genotype seemed to have its own signature MAA composition. These findings indicate a genetic (host, zooxanthellae or both) component to UVR resistance in this population of P. compressa.     

Seasonal plasticity of Ludwigia grandiflora under light and water depth gradients: An outdoor mesocosm experiment

Abiotic factors in the introduced area can limit the establishment of exotic species. Adaptation to new abiotic parameters through plastic responses is important for their establishment and distribution pattern. Survival and growth of exotic hydrophytes are related to their plasticity and their ability to adapt to local conditions. Light and water depth are two of the predominant factors determining survival, growth, distribution and abundance of aquatic plants. Although many studies have been carried out on the effect of water level and light on plant performance, their potential interactions are poorly understood. Here, we carried out an outdoor mesocosm experiment, in order to test the role of water depth, light intensity and their potential interactions on the growth and vigour of the invasive macrophyte species Ludwigia grandiflora. Three light intensities (100%, 70% and 50% of sunlight) and three water depths (30, 50 and 80 cm) were tested over a 6-week period, in spring, summer and autumn 2011. We showed that the mortality, biomass, apical and lateral growth of L. grandiflora, as well as its biomass allocation, varied according to the season and responded to both light intensity and water depth. Interactions between water depth and light intensity can have an amplifying effect on the growth of L. grandiflora. Similarities between shade and deep water adaptation responses were observed. A higher biomass and a high morphological plasticity in response to these factors were found particularly at the beginning of the life cycle. Although the invasive amphibious L. grandiflora shows a high tolerance to different water levels and light intensities, the optimal growth conditions for L. grandiflora in our experiment seem to be under full sunlight and in 30 cm of water. Colonization of light or low shade aquatic habitats, as well as shallow environments, can occur due to the high plasticity of L. grandiflora. These results may be important relative to increasing level of water bodies under climatic changes.     

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.