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Localized transfection on arrays of magnetic beads coated with PCR products.

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Animals↗

Structure of a tumor associated antigen containing a tandemly repeated immunodominant epitope.

Human mucins are T or S glycosylated tandem repeat proteins. In breast cancer, mucins become under or unglycosylated. Two-dimensional nuclear magnetic resonance experiments are performed on chemically synthesized mucin tandem repeat polypeptides, (PDTRPAPGST-APPAHGVTSA)n the unglycosylated form for n=1,3 where (APDTR) constitutes the antigenic sites for the antibodies isolated form the tumors in the breast cancer patients. These studies demonstrate how the tandem repeats assemble in space giving rise to the overall tertiary structure, and the local structure and presentation of the antigenic site(APDTR) at the junction of two neighboring repeats. The NMR data reveal repeating knob-like structures connected by extended spacers. The knobs protrude away from the long-axis of Muc-1 and the predominant antigenic site (APDTR) forms the accessible tip of the knob. Multiple tandem repeats enhance the rigidity and presentation of the knob-like structures.

Amino Acid Sequence↗

LinearCapR: linear-time computation of per-nucleotide structural-context probabilities of RNA without base-pair span limits.

MOTIVATION: RNA molecules adopt dynamic ensembles of secondary structures, where the local structural context of each nucleotide-such as whether it resides in a stem or a specific type of loop-strongly shapes molecular interactions and regulatory function. Structural-context probabilities therefore provide a more functionally informative view of RNA folding than the minimum free energy structures or base-pairing probabilities. However, existing tools either require O(N3) time or employ span-restricted approximations that omit long-range base-pairs, limiting their applicability to large and biologically important RNAs. RESULTS: We introduce LinearCapR, enabling linear-time, span-unrestricted computation of structural-context marginalized probabilities, using beam-pruned Stochastic Context Free Grammar-based computation. LinearCapR retains global ensemble features lost by span-limited methods and yields superior predictive power on bpRNA-1m(90) dataset, especially for multiloops and exterior regions, as well as long-distance stems. LinearCapR supports analysis of long RNAs, demonstrated on the full genome of SARS-CoV-2. LinearCapR provides the first base-pair-span-unrestricted, linear-time framework for RNA structural-context analysis, retaining key thermodynamic ensemble features essential for functional interpretation. It enables large-scale studies of viral genomes, long non-coding RNAs, and downstream analyses such as RNA-binding protein site prediction. AVAILABILITY AND IMPLEMENTATION: The source code of LinearCapR is available at https://github.com/hoget157/LinearCapR. The archived software release used in this work is available at Zenodo: https://doi.org/10.5281/zenodo.19450645.

Nucleic Acid Conformation↗

RNA-related tools on the Bielefeld Bioinformatics Server.

We present four tools for the analysis of RNA secondary structure. They provide animated visualization of multiple structures, prediction of potential conformational switching, structure comparison (including local structure alignment) and prediction of structures potentially containing a certain kind of pseudoknots. All are available via the Bielefeld University Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de).

Base Sequence↗

Elasticity and clustering in concentrated depletion gels.

X-ray scattering and rheology are employed to study the volume fraction dependence of the collective structure and elastic moduli of concentrated nanoparticle-polymer depletion gels. The nonequilibrium gel structure consists of locally densified nonfractal clusters and narrow random interfaces. The elastic moduli display a power law dependence on volume fraction with effective exponents that decrease with increasing depletion attraction strength. A microscopic theory that combines local structural information with a dynamic treatment of gelation is in good agreement with the observations.

Colloids↗

Can the low-resolution structures of photointermediates of bacteriorhodopsin explain their crystal structures?

To understand the molecular mechanism of light-driven proton pumps, the structures of the photointermediates of bacteriorhodopsin have been intensively investigated. Low-resolution diffraction techniques have demonstrated substantial conformational changes at the helix level in the M and N intermediates, between which there are noticeable differences. The intermediate structures at atomic resolution have also been solved by x-ray crystallography. Although the crystal structures have demonstrated local structural changes, such as hydrogen bond network rearrangements including water molecules, the large conformational changes at the helix level are not necessarily observed. Furthermore, the two reported crystal structures of an intermediate accumulated using a common method were distinct. To reconcile these apparent discrepancies, low-resolution projection maps were calculated from the crystal structures and compared to the low-resolution intermediate structures obtained using native membranes. The crystal structures can be categorized into three groups, which qualitatively correspond to the low-resolution structures of the M1-type, M2-type, and N-type determined in the native membrane. Based on these results, we conclude that at least three types of intermediate structures play a role during the photocycle.

Bacteriorhodopsins↗

Heterogeneous cell mechanical properties: an atomic force microscopy study.

Atomic force microscopy (AFM) is a non-invasive microscopy to explore living biological systems like cells in liquid environment. Thus AFM is an appropriate tool to investigate surface chemical modification and its influence on biological systems. In particular, control over biomaterial surface chemistry can result in a regulated cell response. This report investigates the influence of adhesive and non-adhesive surfaces on the cell morphology and the influence of the cytoskeleton structure on the local mechanical properties. In this study, the main work concerns a thorough investigation of the height images obtained with an AFM as therecorded images provide the evolution of the mechanical properties of the cell as function of its local structure. Information on the cell elasticity due to the cytoskeleton organization is deduced when comparing the AFM tip indentation depth versus the distance between the cytoskeleton bundles for the different samples.

Cell Adhesion↗

Interdomain motion in liver alcohol dehydrogenase. Structural and energetic analysis of the hinge bending mode.

A study of the hinge bending mode in the enzyme liver alcohol dehydrogenase is made by use of empirical energy functions. The enzyme is a dimer, with each monomer composed of a coenzyme binding domain and a catalytic domain with a large cleft between the two. Superposition of the apoenzyme and holoenzyme crystal structures is used to determine a rigid rotation axis for closing of the cleft. It is shown that a rigid body transformation of the apoenzyme to the holoenzyme structure corresponds to a 10 degrees rotation of the catalytic domain about this axis. The rotation is not along the least-motion path for closing of the cleft but instead corresponds to the catalytic domain coming closer to the coenzyme binding domain by a sliding motion. Estimation of the energy associated with the interdomain motion of the apoenzyme over a range of 90 degrees (-40 to 50 degrees, where 0 degrees corresponds to the minimized crystal structure) demonstrates that local structural relaxation makes possible large-scale rotations with relatively small energy increments. A variety of structural rearrangements associated with the domain motion are characterized. They involve the hinge region residues that provide the covalent connections between the two domains and certain loop regions that are brought into contact by the rotation. Differences between the energy minimized and the holoenzyme structures point to the existence of alternative conformations for loops and to the importance of the ligands in the structural rearrangements.

Alcohol Dehydrogenase↗

Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae.

The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I-defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.

Cell Nucleolus↗

Electron microscopy of human factor V and factor VIII: correlation of morphology with domain structure and localization of factor V activation fragments.

Clotting factor V and factor VIII are each represented by the domain structure A1-A2-B-A3-C1-C2 and share 40% sequence homology in the A and C domains. Rotary-shadowed samples of human factor V and factor VIII were examined in the electron microscope. Single-chain factor V molecules exhibited a globular "head" domain 12-14 nm in diameter. In addition, up to 25% of these molecules showed a rod-like "tail" of up to 50 nm. Glycerol-gradient centrifugation of factor V treated with thrombin partially resolved the factor Va heterodimer from a larger activation peptide of 150 kDa, as determined by gel electrophoresis. Electron microscopy of factor Va revealed globular molecules with several smaller appendicular structures but lacking the tails seen in factor V. Images of the 150-kDa activation peptide showed rod-like structures, similar in width to the tail of intact factor V and approximately 34 nm long. Rotary shadowing was also used to visualize factor VIII that had been fractionated into heterodimers containing heavy chains of distinct sizes. Each factor VIII preparation showed a globular structure approximately 14 nm in diameter, but the associated tails were observed much more frequently with factor VIII heterodimers containing the higher-molecular-weight heavy chains. These results, in conjunction with results of studies using other biophysical techniques, suggest a model in which the A and C domains of each cofactor constitute a globular head and the connecting B domain is contained in a two-stranded tail that is released by thrombin cleavage.

Electrophoresis, Polyacrylamide Gel↗

A comparison of position-specific score matrices based on sequence and structure alignments.

Sequence comparison methods based on position-specific score matrices (PSSMs) have proven a useful tool for recognition of the divergent members of a protein family and for annotation of functional sites. Here we investigate one of the factors that affects overall performance of PSSMs in a PSI-BLAST search, the algorithm used to construct the seed alignment upon which the PSSM is based. We compare PSSMs based on alignments constructed by global sequence similarity (ClustalW and ClustalW-pairwise), local sequence similarity (BLAST), and local structure similarity (VAST). To assess performance with respect to identification of conserved functional or structural sites, we examine the accuracy of the three-dimensional molecular models predicted by PSSM-sequence alignments. Using the known structures of those sequences as the standard of truth, we find that model accuracy varies with the algorithm used for seed alignment construction in the pattern local-structure (VAST) > local-sequence (BLAST) > global-sequence (ClustalW). Using structural similarity of query and database proteins as the standard of truth, we find that PSSM recognition sensitivity depends primarily on the diversity of the sequences included in the alignment, with an optimum around 30-50% average pairwise identity. We discuss these observations, and suggest a strategy for constructing seed alignments that optimize PSSM-sequence alignment accuracy and recognition sensitivity.

Algorithms↗

Estimating local backbone structural deviation in homology models.

After the atomic coordinates themselves, the most important data in a homology model are the spatial reliability estimates associated with each of the atoms (atom annotation). Recent blind homology modeling predictions have demonstrated that principally correct sequence-structure alignments are achievable to sequence identities as low as 25% [Martin, A.C., MacArthur, M.W., Thornton, J.M., 1997. Assessment of comparative modeling in CASP2. Proteins Suppl(1), 14-28]. The locations and extent of spatial deviations in the backbone between correctly aligned homologous protein structures remained very poorly estimated however, and these errors were the cause of errant loop predictions [Abagyan, R., Batalov, S., Cardozo, T., Totrov, M., Webber, J., Zhou, Y., 1997. Homology modeling with internal coordinate mechanics: deformation zone mapping and improvements of models via conformational search. Proteins Suppl(1), 29-37]. In order to derive accurate measures for local backbone deviations, we made a systematic study of static local backbone deviations between homologous pairs of protein structures. We found that 'through space' proximity to gaps and chain termini, local three-dimensional 'density', three-dimensional environment conservation, and B-factor of the template contribute to local deviations in the backbone in addition to local sequence identity. Based on these finding, we have identified the meaningful ranges of values within which each of these parameters correlates with static local backbone deviation and produced a combined scoring function to greatly improve the estimation of local backbone deviations. The optimized function has more than twice the accuracy of local sequence identity or B-factor alone and was validated in a recent blind structure prediction experiment. This method may be used to evaluate the utility of a preliminary homology model for a particular biological investigation (e.g. drug design) or to provide an improved starting point for molecular mechanics loop prediction methods.

Computer Simulation↗

Cooperative Jahn-Teller phase transition in LaMnO3 studied by X-ray absorption spectroscopy.

The local structure of LaMnO3 across the Jahn-Teller (JT) transition at T(JT)=750 K was studied by means of x-ray absorption near edge structure and extended x-ray absorption fine structure at the Mn K-edge. Our results indicate a similar electronic local structure for Mn atoms above and below T(JT) and a dynamical tetragonal JT distortion of MnO6 octahedra above T(JT). The structural transition is originated by the ordering of tetragonally distorted octahedra. The entropy content of the transition is analyzed within the framework of the three-state Potts model with nearest neighbor antiferrodistortive coupling.

Journal Article↗

Low-resolution docking: prediction of complexes for underdetermined structures.

One of the most fundamental questions concerning ligand-receptor interaction is whether such a process of intermolecular association is generally determined by local structural elements of the participating molecules, or whether there are also large-scale motifs in molecule structures that facilitate complex formation. From the point of view of practical docking computations, the elaborate character of local structural details in ligand-receptor interaction creates a large number of false-positive matches, which interfere with determination of the best fit. Another significant obstacle in protein docking is the problem of structural data inaccuracy (poor structure resolution, conformational changes upon complex formation, etc.). Our study [Vakser (1995) Protein Eng., 8, 371-377], based on ultralow (approximately 7 A resolution) representation of molecular structures, allowes to average all high-resolution structural details, and still predict most of the structural features of the ligand-receptor complex. The approach dramatically improves the signal-to-noise ratio in determination of the best fit, and moves the structure inaccuracy tolerance to the range of the macrostructure. In the present paper, we describe a further validation of the main principles of this approach and a detailed analysis of the low-resolution docking results. This includes clustering of ligand positions around the receptor molecule and cross-validation of ligands and receptors from different complexes. We also discuss the important implications of the approach to the multiple-minima problem and a possible role of different structural elements in the recognition mechanism.

Animals↗

[Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal].

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.

Crystallization↗

Adsorptive properties of metal oxide dispersed carbon materials and characterization of metal oxide fine particles by XAFS.

Surface modification of activated carbon fiber (ACF) with metal oxide increases the adsorptivity of the ACF for supercritical gas such as NO and CH4. In this study, pitch-based activated carbon fibers (P20) were modified with nickel oxide particles. The adsorptive properties and micropore structures of NiO dispersed P20 (NiO-P20) were investigated by nitrogen adsorption isotherms at 77 K. The isotherm was of type 1, suggesting that the NiO-P20 has microporosity. The micropore volume and surface area for NiO-dispersed P20 were smaller than that of P20 by about 15%. The Ni K-edge X-ray absorption fine structure of NiO-P20 has been investigated in order to characterize the species dispersed on P20. The local structure of the NiO particles on the P20 showed some different features compared with the local structure of powdered NiO.

Journal Article↗

Studies of Structure and Local Wetting Properties on Heterogeneous, Micropatterned Solid Surfaces by Microinterferometry.

The microstructure and the local wettability of functionalized patterns is reported. The patterns are formed by the photolithographic structuring of multilayers of hairy rod polymers on glass/MgF2/SiO2 substrates. By application of quantitative reflection interference contrast microscopy (RICM), the RICM image analysis was improved by accounting for (i) multiple reflections and the finite illumination aperture in stratified systems and (ii) geometrical effects in wedge-shaped systems and by (iii) optimizing the contrast enhancement through adjustment of the MgF2 coating. The surface free energy of hairy rod films were determined by analysing the contact angle of partially wetting liquid droplets as a function of the surface tension of the liquid. Periodic lattices of hydrophobic domains separated by hydrophilic domains were applied to check the theory of Joanny and de Gennes [J. Phys. Chem. 81, 552-562 (1984)] of contact angle hysteresis at periodic arrays of pinning centers. Measurements of the shape of the triple line and the contact angle hysteresis by RICM confirmed the theoretical predictions. Copyright 1997 Academic Press.

Journal Article↗

Structure, genetic localization, and identification of the cardiac and skeletal muscle transcripts of the human integrin alpha7 gene (ITGA7).

We have determined the structure and the exon size pattern of the human integrin alpha7 subunit gene (ITGA7), which has been shown to be affected in a form of congenital myopathy. The gene is composed of at least 27 exons spanning a region of about 22.5 kb. The sequence of all exon/intron boundaries was determined and conforms to the GT/AG splicing consensus. We investigated the different splicing forms previously described in human and rodents. The major cytoplasmic variants alpha7A and alpha7B, which are developmentally regulated and tissue specific, were identified in human tissues, as well as the extracellular isoforms X1 and X2. The recently described D variant was detected in adult tissues by RT-PCR but not the C variant. We localized ITGA7 on chromosome 12q13 by high-resolution radiation hybrid mapping between D12S312 and D12S90 and identified a new CA-repeat microsatellite in intron 1.

Amino Acid Sequence↗