Application of 2D and 3D NMR experiments to the conformational study of a diantennary oligosaccharide

Summary By the application of homonuclear 3D NOE-HOHAHA and heteronuclear 3D HMQC-NOE experiments in studies of complex oligosaccharides. NOEs can be investigated which are hidden in conventional 2D NOE spectra. In the 3D NOE-HOHAHA spectrum ₃ cross sections were considered to be the most suitable for assignment of NOEs. Alternatively, these cross sections could be measured separately in selective 2D HOHAHA-NOE spectroscopy. The advantages and limitations of the 2D alternative are compared with those of the 3D NOE-HOHAHA approach. In 3D HMQC-NOE spectroscopy the larger chemical shift displacement of the carbon spectrum with respect to the proton spectrum can be used to unmask NOEs hidden in the bulk region. If the extra proton dimension is not needed, 2D HMQC-NOE is a good alternative.The suitability of 2D and 3D NOE-HOHAHA and HMQC-NOE experiments for the estimation of proton-proton distances is demonstrated by comparing the results of these experiments on a diantennary asparagine-linked oligosaccharide with those of a conventional 2D NOE experiment. NOEs identified in the 2D and 3D NOE-HOHAHA as well as HMQC-NOE experiments, so far not identified or not quantified in 2D NOE experiments, are discussed in relation to each glycosidic linkage. The flexibility of the Man(1-3)Man linkage is demonstrated, confirming the existence of an ensemble of conformations for this linkage.     

Computer-assisted assignment of multidimensional NMR spectra of proteins: Application to 3D NOESY-HMQC and TOCSY-HMQC spectra

Summary An algorithm based on the technique of combinatorial minimization is used for the semi-automated assignment of multidimensional heteronuclear spectra. The program (ALFA) produces the best assignment compatible with the available input data. Even partially misleading or missing data do not seriously corrupt the final assignment. Ambiguous sequences of the possible assignment and all alternatives are indicated. The program can also use additional non-spectroscopic data to assist in the assignment procedure. For example, information from the X-ray structure of the protein and/or information about the secondary structure can be used. The assignment procedure was tested on spectra of mucous trypsin inhibitor, a protein of 107 residues.     

Evolution of the multidomain protein wheat germ agglutinin

Summary We compared the homologous amino acid sequences of hevein and each of the four domains (A, B, C, and D) of wheat germ agglutinin and used them to construct a pseudophylogenetic tree relating these sequences to a hypothetical common ancestor sequence. In the crystal structure of the wheat germ agglutinin dimer, six pseudo-twofold rotational symmetry axes have previously been located in addition to the true twofold axis. Four of these relate two nonidentical domains to each other in each of the four possible pairs constituting the sugar-binding sites (A₁D₂, A₂D₁, B₁C₂, and B₂C₁). The remaining two relate contiguous unique pairs of sugar-binding sites to each other (A₁D₂ to B₁C₂, and A₂D₁ to B₂C₁). These latter two sets of pairs are related to each other by the true twofold axis. Side chains that mediate sugar binding in the interfaces of each of the four pairs were found to be largely conserved. The sequence homology, taken together with these pseudo-symmetry elements in the dimer structure, suggests a pathway for the evolution of the four-domain molecule from a single-domain dimer that can be correlated with simultaneous development of the saccharide-binding sites.     

Phylogeny of a Growth Hormone-Like Cytokine Superfamily Based upon 3D Structure

Abstract Cytokines are of central importance in the regulation of hematopoiesis, immunity, inflammation, tissue remodeling, and embryonic development. Cytokine research is expected to provide the key to pharmacological manipulation of the immune response and commands the attention of a massive and highly focused biotechnology industry. Based upon the hypothetical secondary and tertiary structures, a superfamily of growth hormone (GH)-like cytokine was identified previously. Here, we report the phylogeny of this superfamily based upon 3D structural data from the Protein Data Bank. First, a retrieving program is designed to abstract their secondary structures and associated atomic coordinates. Helices, digitized as vectors in the Cartesian coordinate system, are collected from the retrieved atomic coordinates at the α carbons of the protein backbone. Then the scalar value and vector angle against the reference vector, usually the first vector, are calculated. Furthermore, cluster analysis among various cytokines is performed on their helical scales and helical angles. As a result, GH is close to the cluster formed by ciliary neurotrophic factor and granulocyte colony-stimulating factor (CSF); leptin and erythropoietin are in descending order close to the cluster formed by interleukin (IL)-6 and IL-10; the former seven members in the two subgroups above join together and form one group with leukemia inhibitory factor; granulocyte–macrophage CSF, IL-2, IL-4, and IL-5 are in descending order close to the cluster formed by IL-3 and macrophage CSF; and the latter six members form another group. Finally, it is demonstrated that the phylogeny of GH-like cytokines above is consistent with the evolutionary relationship of their gene organization, gene localization, receptor module composition, and receptor module compatibility.     

Library of disordered patterns in 3D protein structures

Intrinsically disordered regions serve as molecular recognition elements, which play an important role in the control of many cellular processes and signaling pathways. It is useful to be able to predict positions of disordered regions in protein chains. The statistical analysis of disordered residues was done considering 34,464 unique protein chains taken from the PDB database. In this database, 4.95% of residues are disordered (i.e. invisible in X-ray structures). The statistics were obtained separately for the N- and C-termini as well as for the central part of the protein chain. It has been shown that frequencies of occurrence of disordered residues of 20 types at the termini of protein chains differ from the ones in the middle part of the protein chain. Our systematic analysis of disordered regions in PDB revealed 109 disordered patterns of different lengths. Each of them has disordered occurrences in at least five protein chains with identity less than 20%. The vast majority of all occurrences of each disordered pattern are disordered. This allows one to use the library of disordered patterns for predicting the status of a residue of a given protein to be ordered or disordered. We analyzed the occurrence of the selected patterns in three eukaryotic and three bacterial proteomes.     

Population structure in the Connecticut Valley: II. A comparison of multidimensional scaling solutions of migration matrices and isonymy

In a previous paper (Swedlund et al., 1984) we have described the population structure of the historical Connecticut River Valley of Massachusetts in terms of matrimonial migration matrices. Using procedures described by Morton (1973), Harpending and Jenkins (1974), Jorde (1980), and others the exchanges between subdivisions which make up the matrices are made column stochastic and analyzed to predict genetic kinship. Subsequently the kinship estimates within and between subdivisions can be interpreted as genetic covariance and compared to the actual geographic distances between the respective subdivisions using a principal components analysis. In the present paper we extend these results by applying nonmetric multidimensional scaling to the migration matrices, and to isonymy matrices based on the same communities. We demonstrate that the multidimensional scaling configurations of marital migration represent the actual geographic relationships between the communities quite effectively for this particular case study from historical Massachusetts. Moreover, we argue that while these migration data may provide good estimates of social and genetic exchange between the subdivisions, surname analysis may also be informative of processes not revealed in the migration matrices alone.     

Application of a directed conformational search for generating 3-D coordinates for protein structures from alpha-carbon coordinates.

A directed conformational search algorithm using the program CONGEN (ref. 3), which samples backbone conformers, is described. The search technique uses information from the partially built structures to direct the search process and is tested on the problem of generating a full set of backbone Cartesian coordinates given only alpha-carbon coordinates. The method has been tested on six proteins of known structure, varying in size and classification, and was able to generate the original backbone coordinates with RMSs ranging from 0.30-0.87A for the alpha-carbons and 0.5-0.99A RMSs for the backbone atoms. Cis peptide linkages were also correctly identified. The procedure was also applied to two proteins available with only alpha-carbon coordinates in the Brookhaven Protein Data Bank; thioredoxin (SRX) and triacylglycerol acylhydrolase (TGL). All-atom models are proposed for the backbone of both these proteins. In addition, the technique was applied to randomized coordinates of flavodoxin to assess the effects of irregularities in the data on the final RMS. This study represents the first time a deterministic conformational search was used on such a large scale.     

Reconstruction of 3D structures from protein contact maps

The prediction of the protein tertiary structure from solely its residue sequence (the so called Protein Folding Problem) is one of the most challenging problems in Structural Bioinformatics. We focus on the protein residue contact map. When this map is assigned it is possible to reconstruct the 3D structure of the protein backbone. The general problem of recovering a set of 3D coordinates consistent with some given contact map is known as a unit-disk-graph realization problem and it has been recently proven to be NP-Hard. In this paper we describe a heuristic method (COMAR) that is able to reconstruct with an unprecedented rate (3-15 seconds) a 3D model that exactly matches the target contact map of a protein. Working with a non-redundant set of 1760 proteins, we find that the scoring efficiency of finding a 3D model very close to the protein native structure depends on the threshold value adopted to compute the protein residue contact map. Contact maps whose threshold values range from 10 to 18 Ångstroms allow reconstructing 3D models that are very similar to the proteins native structure.     

ProSAT: functional annotation of protein 3D structures

ProSAT (for Protein Structure Annotation Tool) is a tool to facilitate interactive visualization of non-structure-based functional annotations in protein 3D structures. It performs automated mapping of the functional annotations onto the protein structure and allows functional sites to be readily identified upon visualization. The current version of ProSAT can be applied to large datasets of protein structures for fast visual identification of active and other functional sites derived from the SwissProt and Prosite databases.     

Isotope-edited multidimensional NMR of calcineurin B in the presence of the non-deuterated detergent CHAPS

Summary At the concentration needed for NMR, the calcium-saturated form of calcineurin B dissolved in water shows resonance line widths that indicate aggregation of this protein. Although the line width or aggregation state can be influenced to some degree by temperature, pH, and salt concentrations, in the absence of detergent no conditions could be found where the protein behaved as a monomeric unit. In the presence of a 10- to 20-fold molar excess of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), resonance line widths were considerably narrower and were compatible with a protein of 25 kDa. The presence of the NMR signals of the non-deuterated CHAPS does not interfere with modern isotope-directed NMR studies as the signals from protons not attached to ¹⁵N or ¹³C are removed by isotope filtering and purge pulses.     

Pre-docking filter for protein and ligand 3D structures

Virtual drug screening using protein-ligand docking techniques is a time-consuming process, which requires high computational power for binding affinity calculation. There are millions of chemical compounds available for docking. Eliminating compounds that are unlikely to exhibit high binding affinity from the screening set should speed-up the virtual drug screening procedure. We performed docking of 6353 ligands against twenty-one protein X-ray crystal structures. The docked ligands were ranked according to their calculated binding affinities, from which the top five hundred and the bottom five hundred were selected. We found that the volume and number of rotatable bonds of the top five hundred docked ligands are similar to those found in the crystal structures and corresponded with the volume of the binding sites. In contrast, the bottom five hundred set contains ligands that are either too large to enter the binding site, or too small to bind with high specificity and affinity to the binding site. A pre-docking filter that takes into account shapes and volumes of the binding sites as well as ligand volumes and flexibilities can filter out low binding affinity ligands from the screening sets. Thus, the virtual drug screening procedure speed is increased.     

Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G

Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using ¹⁵N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R ₂ values in the -helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T ₁ values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R ₂/R ₁ is anisotropic (D _par/D _perp=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with ¹⁵N relaxation rates and the estimation of R _ex terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.     

Efficient shape descriptors for feature extraction in 3D protein structures

Structural Genomics initiatives are generating an increasing number of protein structures with very limited biochemical characterization. Characterization of a protein's function and understanding the specific nature of a protein's binding is a critical part of both protein engineering and structure-based drug discovery. The accurate detection of binding site in these protein structures can be valuable in determining its function. As shape plays a crucial role in bimolecular recognition and function, the development of shape analysis techniques is important for understanding protein structure-function relationships. This paper describes the use of the continuous wavelet transforms (CWT) for characterizing shape features of 3D protein structures. The goal is to explore the CWT as a multiscale tool to generate rotation- and translation-invariant shape features.     

Generation of native-like protein structures from limited NMR data,modern force fields and advanced conformational sampling

Determining an accurate initial native-like protein fold is one of the most important and time-consuming steps of de novo NMR structure determination. Here we demonstrate that high-quality native-like models can be rapidly generated from initial structures obtained using limited NOE assignments, through replica exchange molecular dynamics refinement with a generalized Born implicit solvent (REX/GB). Conventional structure calculations using an initial sparse NOE set were unable to identify a unique topology for the zinc-bound C-terminal domain of E. coli chaperone Hsp33, due to a lack of unambiguous long range NOEs. An accurate overall topology was eventually obtained through laborious hand identification of long range NOEs. However we were able to obtain high-quality models with backbone RMSD values of about 2 Å with respect to the final structures, using REX/GB refinement with the original limited set of initial NOE restraints. These models could then be used to make further assignments of ambiguous NOEs and thereby speed up the structure determination process. The ability to calculate accurate starting structures from the limited unambiguous NOE set available at the beginning of a structure calculation offers the potential of a much more rapid and automated process for NMR structure determination. Jianhan Chen: Authors contributed equally to this work.Hyung-Sik Won: Authors contributed equally to this work.     

Determinants of slowed diffusion in the complex space of the cerebellar glomerulus

Using analytical solutions for two- and three-dimensional (2D and 3D, respectively) and fractal 2D/3D geometry with an absorbing boundary, we modeled glutamate diffusion in a glomerulus, the structure situated around the mossy fiber (MF) terminal in the cerebellum and surrounded by a glial sheath. The model with fractal geometry gave the best fit of experimental AMPA and NMDA receptor-mediated evoked postsynaptic currents (EPSC) at the MF-granule cell synapse. A comparison of the numerically integrated glutamate concentration in an idealized model of glomerulus morphology with analytical solutions reveals that the peculiarities of glomerulus geometry can explain the better fit by the solution with fractal dimension only of experimental EPSC arising from local release, but not from spillover of glutamate. An asynchronous vesicle release only slightly influences the shape of the spillover waveform. Anomalously slow diffusion of glutamate can be an explanation of the observed discrepancy between experimental results and simulations with the 3D model. A good fit of spillover-induced EPSC obtained in the simulations that use a solution for fractional Brownian motion and a match of experimental estimations and theoretical parameters of the diffusion model confirms this assumption.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 418–431, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

3D model-based approach to identification of laminar structures of the cerebral cortex: Application to Brodmann areas 17 and 18

The human cerebral cortex may be subdivided into architectonic fields according to variations within its laminar structure. Studies have shown correspondences between the locations of functional activation foci and architectonic regions. In order to perform accurate localization of functional activation foci to architectonic regions, a parcellation algorithm capable of segmenting architectonic regions on in vivo imaging datasets is required. This paper presents a novel 3D model-based approach to directly detect cortical layers and classify architectonic fields. The column-like structure of the cortex is modeled using a Laplace equation method which generates a collection of intensity profiles that span the cortical mantle. Bayesian evidence for intensity profile elements belonging to hyper- or hypo-intense bands, which represent cell or myelin poor or rich layers in imaging data, is gathered. A non-isotropic Markov Random Field model is used to encourage contiguous bands as well as a penalty term that completes bands across highly curved cortical regions where neighbouring evidence for banding is strong. This algorithm is validated on a 3D histological dataset of a macaque brain with visible layering at intermediate resolution between high-resolution MRI and histology. The algorithm detects the myelin-rich Stria of Gennari and uses this as the basis for finding the Brodmann Area 17/18 boundary.     

LGA: A method for finding 3D similarities in protein structures

We present the LGA (Local-Global Alignment) method, designed to facilitate the comparison of protein structures or fragments of protein structures in sequence dependent and sequence independent modes. The LGA structure alignment program is available as an online service at http://PredictionCenter.llnl.gov/local/lga. Data generated by LGA can be successfully used in a scoring function to rank the level of similarity between two structures and to allow structure classification when many proteins are being analyzed. LGA also allows the clustering of similar fragments of protein structures.