Cytoscape: a software environment for integrated models of biomolecular interaction networks

Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.     

The relative energetic contributions of dominant P1 pocket versus hydrogen bonding interactions to peptide:class II stability: implications for the mechanism of DM function

Peptides are bound to MHC class II molecules by an array of hydrogen bonds between conserved MHC class II protein side-chains and the peptide backbone and through interactions between MHC protein pockets and peptide side-chain anchors. The crystal structure of murine I-A(k) protein with peptide shows a network of electrostatic interactions with the P1 aspartic acid anchor and an arginine in the P1 pocket that are thought to constitute the major stabilizing interaction between peptide and MHC. In this paper, have explored the relative energetic contribution of this dominant P1 pocket interaction with that made by a genetically conserved hydrogen bond which is formed by the beta 81 histidine residue and the main chain of the bound peptide. We have performed peptide dissociation experiments using antigenic peptides or variants that have altered side-chain interactions with the I-A(k) P1 pocket using either native I-A(k) or I-A(k) proteins mutated to disrupt the N-terminal hydrogen bond. The results demonstrate that the N-terminal hydrogen bonds in I-A(k) complexes make highly significant energetic contributions to the kinetic stabilities comparable to or greater than the energetic contribution of highly favorable P1 pocket interactions. Hence, we conclude that the kinetic stability of MHC class II:peptide complexes critically depends on two quite distinct molecular interactions between peptide and MHC located at the peptide's amino terminus. We discuss these results in light of the proposed mechanism for DM function.     

Kinetic and energetic parameters of imipramine binding to monoclonal antibodies as measured by fluorescence spectroscopy.

Monoclonal antibodies which bind small drugs are useful for the study of the interactive forces involved in antibody-ligand complexation. Detailed understanding of these supramolecular forces requires a careful examination of structural and thermodynamic parameters of the interacting molecules. Fluorescence spectroscopy techniques are very useful in this regard. We report here, the kinetic and energetic parameters of four monoclonal antibodies made against the tricyclic antidepressant imipramine. These monoclonal antibodies were found to possess high to very high binding affinity constants, ranging from 10(7) to 10(10) M-1, and caused fluorescence quenching or enhancement of a fluorescein labelled imipramine. The dissociation rates of the fluorescent ligand from the complexes were measured at different temperatures in order to provide some insight regarding the kinetic and energetic (thermodynamic) parameters of the antibody-ligand binding interactions.     

Thermodynamic analysis of degenerate recognition by the NKG2D immunoreceptor: not induced fit but rigid adaptation

The homodimeric immunoreceptor NKG2D drives the activation of effector cells following engagement of diverse, conditionally expressed MHC class I-like protein ligands. NKG2D recognition is highly degenerate in that a single surface on receptor monomers binds pairs of distinct surfaces on each structurally divergent ligand, simultaneously accommodating multiple nonconservative ligand allelic or isoform substitutions. In contrast to TCR-pMHC and other NK receptor-ligand interactions, thermodynamic and kinetic analyses of four NKG2D-ligand pairs (MIC-A*001, MIC-B*005, ULBP1, and RAE-1beta) reported here show that the relative enthalpic and entropic terms, heat capacity, association rates, and activation energy barriers are comparable to typical, rigid protein-protein interactions. Rather than "induced-fit" binding, NKG2D degeneracy is achieved using distinct interaction mechanisms at each rigid interface.     

CD2 and the nature of protein interactions mediating cell-cell recognition

Summary: Rapid progress has recently been made in characterising the structures of leukocyte cell-surface molecules. Detailed analyses of the structure and interactions of CD2 were the first involving a molecule that has not been directly linked to antigen recognition in the manner of antigen receptors or co-receptors. It seems highly likely that the properties of ligand binding by CD2 are relevant to the general mechanisms of cell-cell recognition. As an example of biological recognition, the defining characteristic of cell-cell contact is that it involves che simultaneous interaction of hundreds, if not thousands, of molecules. Affinity and kinetic analyses of ligand binding by CD2 indicated that the protein interactions mediating cell-cell contact, whilst highly specific, are much weaker than initially anticipated, probably due to the requirement that such contacts be easily reversible. Simultaneously, in addressing the mechanism of this mode of recognition, structural and mutational studies focussed on the role of charged residues clustered in the ligand-binding face of CD2, yielding the concept that electrostatic complementarity, rather than surface-shape complementarity, is the dominant feature of specific, low-affinity protein recognition at die cell surface by CD2. The crystallographic analysis of the CD2-binding domain of CD58 strongly supports this concept.     

Quantitative functional analysis of protein complexes on surfaces

A major challenge in cell and molecular physiology research is to understand the mechanisms of biological processes in terms of the interactions, activities and regulation of the underlying proteins. Functional and mechanistic analyses of the large number of proteins that participate in the regulation of cellular processes will require new approaches and techniques for high throughput and multiplexed functional analyses of protein interactions, protein conformational dynamics and protein activity. In this review we focus on the development and application of proteomics and associated technologies for quantitative functional analysis of proteins and their complexes that include: (1) the application of surface plasmon resonance (SPR) imaging for multiplexed, label-free analyses of protein interactions, binding constants for biomolecular interactions and protein activities; and (2) high content analysis of protein motions within functional multiprotein complexes.     

SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics

Surface plasmon resonance (SPR) sensing has long been used to study biomolecular binding events and their kinetics in a label-free way. This approach has recently been extended to SPR microscopy, which is an ideal tool for probing large microarrays of biomolecules for their binding interactions with various partners and the kinetics of such binding. Commercial SPR microscopes now make it possible to simultaneously monitor binding kinetics on >1300 spots within a protein microarray with a detection limit of approximately 0.3 ng/cm(2), or <50 fg per spot (<1 million protein molecules) with a time resolution of 1s, and spot-to-spot reproducibility within a few percent. Such instruments should be capable of high-throughput kinetic studies of the binding of small ( approximately 200 Da) ligands onto large protein microarrays. The method is label free and uses orders of magnitude less of the precious biomolecules than standard SPR sensing. It also gives the absolute bound amount and binding stoichiometry.     

Earth's orbital chirality and driving force of biomolecular evolution

In our recent studies, it has been suggested that both symmetry breaking (violation) and biological rhythms could be caused by the natural rhythmic right-handed helical force field produced by the Earth's orbital chirality (EOC) (1--3). In this essay, according to the further theoretical and experimental studies, it was suggested that the natural EOC force field could serve as the diving force of biomolecular evolution via the chiral interactions. In addition, the following suggestions also were pointed out: (1) The EOC force field could cause the origin of non-coding repetitive DNA sequences ('Junk DNA') to increase the genomes stability and complexity; (2) The EOC force field could increase the structural order of biological systems via the biomolecular EOC stabilization energy effects; (3) The biological information could be spontaneously produced by the chiral interactions of the protogenes with the EOC force field.     

Transition towards antigen-binding promiscuity of a monospecific antibody

Polyspecificity is defined as the ability of a given antibody molecule to bind a large panel of structurally diverse antigens. A fraction of circulating IgG in all healthy individuals acquires promiscuous antigen-binding activity only after a transient exposure to certain protein destabilizing factors. The molecular mechanisms of this phenomenon are not well understood. Exposures to protein destabilizing agents are common steps in immunoglobulin isolation and purification processes. We performed kinetic and thermodynamic analyses using surface plasmon resonance-based technique in order to characterize the interactions of a single mouse monoclonal antibody to its cognate antigen before and after induction of promiscuous antigen-binding activity. The obtained results, suggest that enhanced antigen binding activity induced by exposure to mild denaturing condition resulted from an increase in the structural flexibility of the antigen-binding site. Further pH and ionic strength-dependence analyses of the antibody/antigen interactions demonstrated that the transition to promiscuous antigen-binding was accompanied by a change in the type of non-covalent forces involved in the complex formation. Moreover, from this study, it is evident that an antibody molecule could use two distinct thermodynamic pathways for binding to the same antigen while retaining the same value of the binding affinity. The obtained results may contribute to the understanding of the molecular mechanisms that lay behind natural antibody polyspecificity.     

Rapid Microfluidic Perfusion Enabling Kinetic Studies of Lipid Ion Channels in a Bilayer Lipid Membrane Chip

There is growing recognition that lipids play key roles in ion channel physiology, both through the dynamic formation and dissolution of lipid ion channels and by indirect regulation of protein ion channels. Because existing technologies cannot rapidly modulate the local (bio)chemical conditions at artificial bilayer lipid membranes used in ion channel studies, the ability to elucidate the dynamics of these lipid–lipid and lipid–protein interactions has been limited. Here we demonstrate a microfluidic system supporting exceptionally rapid perfusion of reagents to an on-chip bilayer lipid membrane, enabling the responses of lipid ion channels to dynamic changes in membrane boundary conditions to be probed. The thermoplastic microfluidic system allows initial perfusion of reagents to the membrane in less than 1 s, and enables kinetic behaviors with time constants below 10 s to be directly measured. Application of the platform is demonstrated toward kinetic studies of ceramide, a biologically important lipid known to self-assemble into transmembrane ion channels, in response to dynamic treatments of small ions (La³⁺) and proteins (Bcl-x_L mutant). The results reveal the broader potential of the technology for studies of membrane biophysics, including lipid ion channel dynamics, lipid–protein interactions, and the regulation of protein ion channels by lipid micro domains.     

Bioassays based on molecular nanomechanics

Recent experiments have shown that when specific biomolecular interactions are confined to one surface of a microcantilever beam, changes in intermolecular nanomechanical forces provide sufficient differential torque to bend the cantilever beam. This has been used to detect single base pair mismatches during DNA hybridization, as well as prostate specific antigen (PSA) at concentrations and conditions that are clinically relevant for prostate cancer diagnosis. Since cantilever motion originates from free energy change induced by specific biomolecular binding, this technique is now offering a common platform for label-free quantitative analysis of protein-protein binding, DNA hybridization DNA-protein interactions, and in general receptor-ligand interactions. Current work is focused on developing "universal microarrays" of microcantilever beams for high-throughput multiplexed bioassays.     

Alginate Films as Macromolecular Imprinted Matrices

Macromolecularly imprinted polymers have been developed to mimic the non-covalent interactions driving molecular recognition in nature. The creation of an engineered antibody mimic would allow for the development of customizable films for biomolecular sensing. To demonstrate this principle, a cross-linked alginate film has been imprinted with bovine serum albumin (BSA) using aqueous biocompatible gelation methods. The imprinting efficiency of the synthesized films imprinted with BSA was determined and compared to the non-specific uptake of complementary proteins which were not imprinted in the alginate matrix. It was found that the recognition of the BSA using an alginate film was 6.4 mg/g polymer, which compares favorably to previously reported macromolecularly imprinted networks. The absorption of non-imprinted cationic proteins by the alginate matrix demonstrates that overcoming non-specific binding needs to be a focus of future work in order to successfully employ these materials towards biomolecular sensing within a physiological environment.     

A theoretical analysis of the thermodynamic contributions for the adsorption of individual protein residues on functionalized surfaces

Although the denaturing of adsorbed proteins on biomaterials surfaces is believed to lead to adverse tissue reactions to implanted materials, very little is currently known of the actual mechanisms involved. These mechanisms must be understood if surfaces are to be proactively designed to control protein adsorption behavior. Concepts widely employed in rational drug design and in protein and RNA folding predictions provide a means to approach this problem. Accordingly, a theoretical analysis has been conducted to estimate the thermodynamic contributions (changes in enthalpy, entropy, and Gibbs free energy) for the adsorption of selected individual mid-chain protein residues to functionalized surfaces. Enthalpic contributions from residue-surface interactions were calculated using semi-empirical quantum mechanical-based computational chemistry methods in a simulated aqueous environment (MOPAC/COSMO), and enthalpic and entropic contributions due to water restructuring effects assumed to occur during adsorption were estimated from experimental data for functional group wetting and calculated changes in solvent accessible surface area as each protein residue approached each surface. When combined with intraprotein residue-residue interactions, the understanding of residue-surface adsorption energy relationships provides a means to begin to predict protein adsorption behavior as a function of biomaterials surface chemistry. It is recognized that several assumptions have been made in this approach that could be challenged, and that truncations necessary due to programming limitations have been applied that may neglect potentially important interactions. Therefore, it must be understood that the modeling predictions may not be directly applicable to biomaterials for surface design under actual physiologic conditions at this stage. However, this attempt at modeling fundamental components of protein adsorption is presented as an initial approach to understanding these complex events.     

Glucoamylase absorption and desorption process

This paper reports the study of glucoamylase absorption and desorption processes on spherical particles constituting acrylic supports. The kinetic (reaction order, half-life of the reaction, reaction rate constant), and thermodynamic parameters (activation energy, pre-exponential factor) of the glucoamylase immobilization reaction dynamics inside the acrylic supports have been studied. Hindrance of the penetrants (protein molecules), which defeats the polymer's cohesive forces and passes through the diffusional barriers towards the sites available to receive it, denotes the influence of the diffusion phenomena, and might be expressed by the values of the effective diffusion coefficient and of the energetic parameters, as well. The desorption dynamics of glucoamylase in specific buffer concentration gradients and temperature are dependent on the eluent volume and on the protein eluate.     

The dependence of DNA supercoiling on solution electrostatics

We develop an elastic-isotropic rod model for twisted DNA in the plectonemic regime. We account for DNA elasticity, electrostatic interactions and entropic effects due to thermal fluctuations. We apply our model to single-molecule experiments on a DNA molecule attached to a substrate at one end, while subjected to a tensile force and twisted by a given number of turns at the other end. The free energy of the DNA molecule is minimized subject to the imposed end rotations. We compute values of the torsional stress, radius, helical angle and key features of the rotation-extension curves. We also include in our model the end loop energetic contributions and obtain estimates for the jumps in the external torque and extension of the DNA molecule seen in experiments. We find that, while the general trends seen in experiments are captured simply by rod mechanics, the details can be accounted for only with the proper choice of electrostatic and entropic interactions. We perform calculations with different ionic concentrations and show that our model yields excellent fits to mechanical data from a large number of experiments. Our methods also allow us to consider scenarios where we have multiple plectonemes or a series of loops forming in the DNA instead of plectonemes. For a given choice of electrostatic and entropic interactions, we find there is a range of forces in which the two regimes can coexist due to thermal motion.     

Uses of Biosensors in the Study of Viral Antigens

The introduction in 1990 of a new biosensor technology based on surface plasmon resonance has greatly simplified the measurement of binding interactions in biology. This new technology known as biomolecular interaction analysis makes it possible to visualize the binding process as a function of time by following the increase in refractive index that occurs when one of the interacting partners binds to its ligand immobilized on the surface of a sensor chip. None of the reactants needs to be labelled, which avoids the artefactual changes in binding properties that often result when the molecules are labelled. Biosensor instruments are well-suited for the rapid mapping of viral epitopes and for identifying which combinations of capturing and detector Mabs will give the best results in sandwich assays. Biosensor binding data are also useful for selecting peptides to be used in diagnostic solid-phase immunoassays. Very small changes in binding affinity can be measured with considerable precision which is a prerequisite for analyzing the functional effect and thermodynamic implications of limited structural changes in interacting molecules. On-rate (k_a) and off-rate (k_d) kinetic constants of the interaction between virus and antibody can be readily measured and the equilibrium affinity constant K can be calculated from the ratio k_a/k_d = K.     

Insights from in situ analysis of TCR–pMHC recognition: response of an interaction network

Recognition of peptide presented by the major histocompatibility complex (pMHC) molecule by the T-cell receptor (TCR) determines T-cell selection, development, differentiation, fate, and function. Despite intensive studies on the structures, thermodynamic properties, kinetic rates, and affinities of TCR–pMHC interactions in the past two decades, questions regarding the functional outcome of these interactions, i.e. how binding of the αβ TCR heterodimer with distinct pMHCs triggers different intracellular signals via the adjacent CD3 components to produce different T-cell responses, remain unclear. Most kinetic measurements have used surface plasmon resonance, a three-dimensional (3D) technique in which fluid-phase receptors and ligands are removed from their cellular environment. Recently, several two-dimensional (2D) techniques have been developed to analyze molecular interactions on live T cells with pMHCs presented by surrogate antigen-presenting cells or supported planar lipid bilayers. The insights from these in situ analyses have provided a sharp contrast of the 2D network biology approach to the 3D reductionist approach and prompted rethinking of our current views of T-cell triggering. Based on these insights, we propose a mechanochemical coupled triggering hypothesis to explain why the in situ kinetic parameters differ so much from their 3D counterparts, yet correlate so much better with T-cell functional responses.