代谢光反应交联技术在生物大分子相互作用研究中的应用

具备光反应交联活性的苯甲酮基团、叠氮苯基团以及双吖丙啶基团被广泛应用于对糖、氨基酸等代谢底物的修饰。利用生物自身的代谢机制,引入带有光反应交联基团的底物类似物,可以实现生物大分子间相互作用的原位、实时研究,从而获得更为准确的相互作用复合物的信息。本文将综述代谢光反应交联技术在聚糖-蛋白质、蛋白质-蛋白质相互作用研究中的应用进展。     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level.

The advent of single-molecule biology has allowed unprecedented insight into the dynamic behavior of biological macromolecules and their complexes. Unexpected properties, masked by the asynchronous behavior of myriads of molecules in bulk experiments, can be revealed; equally importantly, individual members of a molecular population often exhibit distinct features in their properties. Finally, the single-molecule approaches allow us to study the behavior of biological macromolecules under applied tension or torsion; understanding the mechanical properties of these molecules helps us understand how they function in the cell. In this review, we summarize the application of magnetic tweezers (MT) to the study of DNA behavior at the single-molecule level. MT can be conveniently used to stretch DNA and introduce controlled levels of superhelicity into the molecule and to follow to a high definition the action of different types of topoisomerases. Its potential for chromatin studies is also enormous, and we will briefly present our first chromatin results.     

Solute size effects on the diffusion in biofilms of Streptococcus mutans

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.     

Unlocking the secrets to protein–protein interface drug targets using structural mass spectrometry techniques

Protein–protein interactions (PPIs) drive all biologic systems at the subcellular and extracellular level. Changes in the specificity and affinity of these interactions can lead to cellular malfunctions and disease. Consequently, the binding interfaces between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Unfortunately, protein–protein contact points have proven to be very difficult pharmacological targets because they are hidden within complex 3D interfaces. For the vast majority of characterized binary PPIs, the specific amino acid sequence of their close contact regions remains unknown. There has been an important need for an experimental technology that can rapidly reveal the functionally important contact points of native protein complexes in solution. In this review, experimental techniques employing mass spectrometry to explore protein interaction binding sites are discussed. Hydrogen–deuterium exchange, hydroxyl radical footprinting, crosslinking and the newest technology protein painting are compared and contrasted.     

Solute Size Effects on the Diffusion in Biofilms of Streptococcus mutans

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.     

Pleomorphic Ensembles: Formation of Large Clusters Composed of Weakly Interacting Multivalent Molecules

Molecular interactions of importance to cell biology are subject to sol-gel transitions: large clusters of weakly interacting multivalent molecules (gel phase) are produced at a critical concentration of monomers. Examples include cell-cell and cell-matrix adhesions, nucleoprotein bodies, and cell signaling platforms. We use the term pleomorphic ensembles (PEs) to describe these clusters, because they have dynamic compositions and sizes and have rapid turnover of their molecular constituents; this plasticity can be highly responsive to cellular signals. The classical polymer physical chemistry theory developed by Flory and Stockmayer provides a brilliant framework for treating multivalent interactions for simple idealized systems. But the complexity and variability of PEs challenges existing modeling approaches. Here we describe and validate a computational algorithm that extends the Flory-Stockmayer formalism to overcome the limitations of analytic theories. We divide the problem by deterministically calculating the fraction of bound sites for each type of binding site, followed by the stochastic assignment of the bonds to a finite number of molecules. The method allows for high valency within many different kinds of interacting molecules and site types, permits simulation of steady-state distributions, as well as assembly kinetics, and can treat cooperative binding within one of the interacting molecules. We then apply our method to the analysis of interactions in the nephrin-Nck-N-Wasp signaling system, demonstrating how multivalent layered scaffolds produce PEs at low monomer concentrations despite weak binding interactions. We show how the experimental data for this system are most consistent with synergistic cooperative interactions between Nck and N-Wasp.     

Pleomorphic Ensembles: Formation of Large Clusters Composed of Weakly Interacting Multivalent Molecules

Molecular interactions of importance to cell biology are subject to sol-gel transitions: large clusters of weakly interacting multivalent molecules (gel phase) are produced at a critical concentration of monomers. Examples include cell-cell and cell-matrix adhesions, nucleoprotein bodies, and cell signaling platforms. We use the term pleomorphic ensembles (PEs) to describe these clusters, because they have dynamic compositions and sizes and have rapid turnover of their molecular constituents; this plasticity can be highly responsive to cellular signals. The classical polymer physical chemistry theory developed by Flory and Stockmayer provides a brilliant framework for treating multivalent interactions for simple idealized systems. But the complexity and variability of PEs challenges existing modeling approaches. Here we describe and validate a computational algorithm that extends the Flory-Stockmayer formalism to overcome the limitations of analytic theories. We divide the problem by deterministically calculating the fraction of bound sites for each type of binding site, followed by the stochastic assignment of the bonds to a finite number of molecules. The method allows for high valency within many different kinds of interacting molecules and site types, permits simulation of steady-state distributions, as well as assembly kinetics, and can treat cooperative binding within one of the interacting molecules. We then apply our method to the analysis of interactions in the nephrin-Nck-N-Wasp signaling system, demonstrating how multivalent layered scaffolds produce PEs at low monomer concentrations despite weak binding interactions. We show how the experimental data for this system are most consistent with synergistic cooperative interactions between Nck and N-Wasp.     

Van der Waals interactions involving proteins.

Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models, with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.     

Short-range RNA-RNA crosslinking methods to determine rRNA structure and interactions

We describe details of procedures to analyze RNA-RNA crosslinks made by far-UV irradiation (< 300 nm) or made by irradiation with near-UV light (320-365 nm) on RNA containing photosensitive nucleotides, in the present case containing 4-thiouridine. Zero-length crosslinks of these types must occur because of the close proximity of the participants through either specific interactions or transient contacts in the folded RNA structure, so they are valuable monitors of the conformation of the RNA. Procedures to produce crosslinks in the 16S ribosomal RNA and between the 16S rRNA and mRNA or tRNA are described. Gel electrophoresis conditions are described that separate the products according to their structure to allow the determination of the number and frequency of the crosslinking products. Gel electrophoresis together with an ultracentrifugation procedure for the efficient recovery of RNA from the polyacrylamide gels allows the purification of molecules containing different crosslinks. These separation techniques allow the analysis of the sites of crosslinking by primer extension and RNA sequencing techniques. The procedures are applicable to other types of RNA molecules with some differences to control levels of crosslinking and separation conditions.     

Extracellular matrix-cell interactions: Focus on therapeutic applications

Extracellular matrix (ECM) macromolecules together with a multitude of different molecules residing in the extracellular space play a vital role in the regulation of cellular phenotype and behavior. This is achieved via constant reciprocal interactions between the molecules of the ECM and the cells. The ECM-cell interactions are mediated via cell surface receptors either directly or indirectly with co-operative molecules. The ECM is also under perpetual remodeling process influencing cell-signaling pathways on its part. The fragmentation of ECM macromolecules provides even further complexity for the intricate environment of the cells. However, as long as the interactions between the ECM and the cells are in balance, the health of the body is retained. Alternatively, any dysregulation in these interactions can lead to pathological processes and finally to various diseases. Thus, therapeutic applications that are based on retaining normal ECM-cell interactions are highly rationale. Moreover, in the light of the current knowledge, also concurrent multi-targeting of the complex ECM-cell interactions is required for potent pharmacotherapies to be developed in the future.     

Reactive nitrogen species in cellular signaling

The transduction of cellular signals occurs through the modification of target molecules. Most of these modifications are transitory, thus the signal transduction pathways can be tightly regulated. Reactive nitrogen species are a group of compounds with different properties and reactivity. Some reactive nitrogen species are highly reactive and their interaction with macromolecules can lead to permanent modifications, which suggested they were lacking the specificity needed to participate in cell signaling events. However, the perception of reactive nitrogen species as oxidizers of macromolecules leading to general oxidative damage has recently evolved. The concept of redox signaling is now well established for a number of reactive oxygen and nitrogen species. In this context, the post-translational modifications introduced by reactive nitrogen species can be very specific and are active participants in signal transduction pathways. This review addresses the role of these oxidative modifications in the regulation of cell signaling events.     

Determination of the refractive index increments of small molecules for correction of surface plasmon resonance data

The refractive index increments (RIIs) of several important low-molecular-weight compounds that bind to DNA or RNA were determined with a differential refractometer for correction of data obtained on surface plasmon resonance (SPR) biosensors. Although the ability to investigate small molecule-macromolecule interactions by SPR is relatively new, the technique is rapidly becoming a primary method to screen focused combinatorial libraries and to quantitatively characterize the interactions between compounds identified as binders and target macromolecules. The most widely used SPR analysis software, BIAevaluation (Biacore, Inc.), assumes that the RIIs of ligand and macromolecule are identical. While the assumption is reasonable for studies involving like molecules such as protein-protein interactions, results presented here demonstrate that RII values for small molecules can be significantly different than those of protein or nucleic acid receptors. The results also show that the RII values can vary greatly depending on the structure of the small molecule. Indeed, the RIIs of the molecules investigated here differ by a factor of 2. Any difference in the RII of interacting molecules must be considered for complete analysis of SPR data. Failure to correct for RII differences can result in serious error in data interpretation, especially for systems involving a ligand:receptor stoichiometry greater than 1. The results serve as the beginning of an SPR correction database for the RIIs of small molecules. Additionally, the results can be used to approximate the RIIs of a variety of other small molecules.     

Mapping the human protein interactome

Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.     

Capturing native interactions: intrinsic methods to study chromatin conformation

The 3D organization of chromatin controls gene expression through spatial interactions between genomic loci. FISH and 3C‐based methods that are commonly used to study chromatin organization utilize chemical crosslinking, a step that may introduce biases in detectable chromatin interactions. In their recent study, Papantonis and colleagues (Brant et al, 2016 ) developed alternative new methods of detecting chromatin contacts without the use of chemical crosslinking agents. These tools increase the resolution and confidence at which interactions can be identified, and may be informative for chromatin interaction dynamics.     

Long-distance interactions and principles of molecular recognition at various biosystem organization levels

Efficient molecular recognition, in which recognition processes are occurring much faster than it takes to test variants, is only possible when long-distance recognition occurs together with contact interactions. The distance between interacting molecules should be sufficiently long to prevent hindrances to the search and, on the other hand, sufficiently short to provide selectivity. It was demonstrated that both of these two requirements can be satisfied simultaneously for biological macromolecules that include helical segments. Because the “diameters” of helical molecules are far shorter than their lengths, the intermolecular distance can be far greater than the diameters, thus allowing a free search. The distance can be far shorter than the lengths at the same time, thus providing selectivity. Analytical procedures were developed to estimate the parameters for protein–protein and protein–nucleic acid recognition. The coincidence of the charge-distribution periods in helical segments was found to substantially increase their interaction potential, and the reduction scale characteristic of the potential was shown to depend on the numerical value of the coinciding period.     

Using topic modeling to detect cellular crosstalk in scRNA-seq

Cell-cell interactions are vital for numerous biological processes including development, differentiation, and response to inflammation. Currently, most methods for studying interactions on scRNA-seq level are based on curated databases of ligands and receptors. While those methods are useful, they are limited to our current biological knowledge. Recent advances in single cell protocols have allowed for physically interacting cells to be captured, and as such we have the potential to study interactions in a complemantary way without relying on prior knowledge. We introduce a new method based on Latent Dirichlet Allocation (LDA) for detecting genes that change as a result of interaction. We apply our method to synthetic datasets to demonstrate its ability to detect genes that change in an interacting population compared to a reference population. Next, we apply our approach to two datasets of physically interacting cells to identify the genes that change as a result of interaction, examples include adhesion and co-stimulatory molecules which confirm physical interaction between cells. For each dataset we produce a ranking of genes that are changing in subpopulations of the interacting cells. In addition to the genes discussed in the original publications, we highlight further candidates for interaction in the top 100 and 300 ranked genes. Lastly, we apply our method to a dataset generated by a standard droplet-based protocol not designed to capture interacting cells, and discuss its suitability for analysing interactions. We present a method that streamlines detection of interactions and does not require prior clustering and generation of synthetic reference profiles to detect changes in expression.     

Real-time analysis of antibody binding interactions with immobilized E. coli O157:H7 cells using the BIAcore

The kinetic properties of antibody-antigen reaction and other interacting macromolecules can be analyzed in real-time using the surface plasmon resonance biosensor (BIAcore). The interactions of an antibody against Escherischia coli O157:H7 were studied using immobilized whole cells. The bacterial sensor surface was evaluated with anti-E. coli in a continuous flow system. Regeneration of the sensor surface with guanidine-HCl was more effective than with phosphoric acid and resulted in better binding reproducibility. The determined kinetic values, association and dissociation rate constants, can be used in the development of rapid immuno-techniques. This study also provides the basis to evaluate real-time interactions of macromolecules with immobilized cells.