Stretching DNA and RNA to probe their interactions with proteins

When interacting with a single stretched DNA, many proteins modify its end-to-end distance. This distance can be monitored in real time using various micromanipulation techniques that were initially used to determine the elastic properties of bare nucleic acids and their mechanically induced structural transitions. These methods are currently being applied to the study of DNA enzymes such as DNA and RNA polymerases, topoisomerases and structural proteins such as RecA. They permit the measurement of the probability distributions of the rate, processivity, on-time, affinity and efficiency for a large variety of DNA-based molecular motors.     

Immobilization and clustering of structurally defined oligosaccharides for sugar chips: an improved method for surface plasmon resonance analysis of protein-carbohydrate interactions

Oligosaccharides are increasingly being recognized as important partners in receptor-ligand binding and cellular signaling. Surface plasmon resonance (SPR) is a very powerful tool for the real-time study of the specific interactions between biological molecules. We report here an advanced method for the immobilization of oligosaccharides in clustered structures for SPR and their application to the analysis of heparin-protein interactions. Reductive amination reactions and linker molecules were designed and optimized. Using mono-, tri-, or tetravalent linker compounds, we incorporated synthetic structurally defined disaccharide units of heparin and immobilized them as ligands for SPR. Their binding to an important hemostatic protein, von Willebrand factor (vWf), and its known heparin-binding domain was quantitatively analyzed. These multivalent ligand conjugates exhibited reproducible binding behavior, with consistency of the surface conditions of the SPR chip. This novel technique for oligosaccharide immobilization in SPR studies is accurate, specific, and easily applicable to both synthetic and naturally derived oligosaccharides.     

Postexercise protein-carbohydrate and carbohydrate supplements increase muscle glycogen in men and women

Tarnopolsky, M. A., M. Bosman, J. R. MacDonald, D. Vandeputte, J. Martin, and B. D. Roy. Postexerciseprotein-carbohydrate and carbohydrate supplements increase muscleglycogen in men and women. J. Appl.Physiol. 83(6): 1877-1883, 1997.We havepreviously demonstrated that women did not increase intramuscularglycogen in response to an increased percent of dietary carbohydrate(CHO) (from 60 to 75% of energy intake) (M. A. Tarnopolsky, S. A. Atkinson, S. M. Phillips, and J. D. MacDougall.J. Appl. Physiol. 78: 1360-1368, 1995). CHO and CHO-protein (Pro) supplementation postexercise canpotentiate glycogen resynthesis compared with placebo (K. M. Zawadzki,B. B. Yaspelkis, and J. L. Ivy. J. Appl.Physiol. 72: 1854-1859, 1992). We studied theeffect of isoenergetic CHO and CHO-Pro-Fat supplements on muscleglycogen resynthesis in the first 4 h after endurance exercise (90 minat 65% peak O₂ consumption) intrained endurance athletes (men, n = 8; women, tested in midfollicular phase,n = 8). Each subject completed threesequential trials separated by 3 wk; a supplement was provided immediately and 1-h postexercise: 1)CHO (0.75 g/kg) + Pro (0.1 g/kg) + Fat (0.02 g/kg),2) CHO (1 g/kg), and3) placebo (Pl; artificialsweetener). Subjects were given prepackaged, isoenergetic, isonitrogenous diets, individualized to their habitual diet, for theday before and during the exercise trial. During exercise, womenoxidized more lipid than did men (P < 0.05). Both of the supplement trials resulted in greaterpostexercise glucose and insulin compared with Pl(P < 0.01), with no genderdifferences. Similarly, both of these trials resulted in increasedglycogen resynthesis (37.2 vs. 24.6 mmol · kg drymuscle¹ · h¹,CHO vs. CHO-Pro-Fat, respectively) compared with Pl (7.5 mmol · kg drymuscle¹ · h¹;P < 0.001) with no genderdifferences. We conclude that postexercise CHO and CHO-Pro-Fatnutritional supplements can increase glycogen resynthesis to a greaterextent than Pl for both men and women.

     

NMR spectroscopic and molecular modeling studies of protein-carbohydrate and protein-peptide interactions

Investigations of the conformations of carbohydrates, their analogues and their molecular mimics are described, with emphasis on structural and functional information that can be gained by NMR spectroscopic techniques in combination with molecular modeling. The transferred nuclear Overhauser effect (trNOE) has been employed to determine the bound conformations of carbohydrates and other bioactive molecules in complex with protein receptors. The corresponding experiments in the rotating frame (trROE) and selective editing experiments (e.g., QUIET-NOESY) are used to eliminate indirect cross-relaxation pathways (spin diffusion), thereby minimizing errors in the data used for calculation of conformations. Saturation transfer difference NMR experiments reveal detailed information about intermolecular contacts between ligand and protein. Computational techniques are integrated with NMR-derived information to construct structural models of these bioactive molecules and of their complexes with proteins. Recent investigations into the nature of molecular mimicry with regard to protein-ligand interactions are described, along with applications in determining the mode of action of enzyme inhibitors. The results are relevant for the design of the next generation of drug and vaccine candidates.     

Kinetics of protein-nucleic acid interactions: use of salt effects to probe mechanisms of interaction

The kinetics of protein-nucleic acid interactions are discussed with particular emphasis on the effects of salt concentration and valence on the observed rate constants. A general review is given of the use of experimentally determined salt dependences of observed kinetic parameters as a tool to probe the mechanism of interaction. Quantitative analysis of these salt dependences, through the application of polyelectrolyte theory, can be used to distinguish reactions which occur in a single step from those reactions which involve distinct intermediates. For those rate constants which display a large salt dependence, in either the association or dissociation reaction, this is due to the high concentration of counterions (e.g., Na+) in the vicinity of the nucleic acid which are subsequently released (or bound in the case of dissociation) at some point before the rate limiting step of the reaction. A general discussion of other features which affect protein-nucleic acid kinetics, such as nucleic acid length and the ratio of nonspecific to specific DNA binding sites (in the case of sequence specific binding proteins), is also given. The available data on the nucleic acid binding kinetics of small ligands (ions, dyes, oligopeptides), nonspecific binding proteins (T4 gene 32 protein, fd gene 5 and Escherichia coli SSB), and sequence specific binding proteins (lac repressor, RNA polymerase, Eco RI restriction endonuclease) are discussed with emphasis on the interpretation of the experimentally determined salt dependences.     

Multivalent protein-carbohydrate interactions. A new paradigm for supermolecular assembly and signal transduction

Many biological recognition processes involve the binding and clustering of ligand-receptor complexes and concomitant signal transduction events. Such interactions have recently been observed in human T cells in which binding and cross-linking of specific glycoprotein counter-receptors on the surface of the cells by an endogenous bivalent carbohydrate binding protein (galectin-1) leads to apoptosis [Pace, K. E., et al. (1999) J. Immunol. 163, 3801-3811]. Importantly, different counter-receptors associated with specific phosphatase or kinase activities were shown to form separate clusters on the surface of the cells as a result of galectin-1 binding to the carbohydrate moieties of the respective glycoproteins. This suggests that the unique separation and organization of signaling molecules that results from galectin-1 binding is involved in delivering the signal to die. The ability of galectin-1 to induce the separation of specific glycoprotein receptors was modeled on the basis of molecular and structural studies of the binding of multivalent carbohydrates to lectins that result in the formation of specific two- and three-dimensional cross-linked lattices. These latter studies have been recently highlighted by X-ray crystallographic results showing that a single tetravalent lectin forms distinct cross-linked complexes with four different bivalent oligosaccharides [Olsen, L. R., et al. (1997) Biochemistry 36, 15073-15080]. In this report, binding and cross-linking of multivalent carbohydrates with multivalent lectins is shown to be a new paradigm for supermolecular assembly and signal transduction in biological systems.     

Reassessing the role of protein-carbohydrate complementarity during sperm-egg interactions in the mouse

Despite years of intense study by many investigators, it may appear that we have made little progress towards a molecular understanding of mammalian sperm binding to the egg zona pellucida. An abundance of evidence derived from in vitro assays suggests that sperm-zona pellucida binding is dependent upon sperm recognition of specific glycan moieties on the zona pellucida glycoproteins. However, there is considerable disagreement regarding the identity of the zona pellucida sugars thought to mediate sperm binding, as well as disagreement over the identity of the sperm receptors themselves. Moreover, results from in vivo gene-targeting strategies fail to support a role for many, if not all, of the sperm receptors and their zona pellucida ligands implicated from in vitro assays. Nevertheless, a retrospective view of the literature suggests that some common principles are emerging regarding the molecular basis of mammalian sperm-zona binding, both with respect to the nature of the components that mediate binding, as well as the involvement of distinct receptor-ligand interactions, that involve both protein- and carbohydrate-dependent mechanisms of binding.     

Characteristics of protein-carbohydrate interactions as a basis for developing novel carbohydrate-based antirejection therapies

The relative shortage of human organs for transplantation is today the major barrier to a broader use of transplantation as a means of treating patients with end-stage organ failure. This barrier could be partly overcome by an increased use of blood group ABO-incompatible live donors, and such trials are currently underway at several transplant centres. If xenotransplantation can be used clinically in the future, the human organ shortage will, in principle, be eradicated. In both these cases, carbohydrate antigens and the corresponding anti-carbohydrate antibodies are the major primary immunological barriers to overcome. Refined carbohydrate-based therapeutics may permit an increased number of ABO-incompatible transplantations to be carried out, and may remove the initial barriers to clinical xenotransplantation. Here, we will discuss the chemical characteristics of protein-carbohydrate interactions and outline carbohydrate-based antirejection therapies as used today in experimental as well as in clinical settings. Novel mucin-based adsorbers of natural anti-carbohydrate antibodies will also be described.     

Conformation and intermolecular interactions of carbohydrate chains

For consideration of their conformations and interactions, carbohydrate chains can conveniently be divided into 3 classes on the basis of their covalent structure; namely periodic (a), interrupted periodic (b), and aperiodic (c) types. In aqueous solution carbohydrate chains often exist as highly disordered random coils. Under appropriate conditions, however, polysaccharides of types (a) and (b) can adopt a variety of ordered conformations. Physical methods, and in particular optical rotation, circular dichroism, and nuclear magnetic resonance, provide sensitive probes for the study of the mechanism and specificity of these disorder-order transitions in aqueous solution. Intermolecular interactions between such polysaccharide chains arise from co-operative associations of long structurally regular regions which adopt the ordered conformations. For acidic polysaccharides these cooperative associations may involve alignment of extended ribbons with cations sandwhiched between them. In other systems the interactions involve double belices which may then aggregate further, and geometric “matching” of different polysaccharide chains can also occur. These ordered, associated regions are generally terminated by deviations from structural regularity or by “kinks” which prevent complete aggregation of the molecules. The complex carbohydrate chains which occur at the periphery of animal cells have very different, aperiodic structures and although their conformations are as yet poorly understood, preliminary indications are considered.     

Plant-microbe interactions to probe regulation of plant carbon metabolism

Plant growth and development is dependent on coordinated assimilate production, distribution and allocation. Application of biochemical and molecular techniques substantially contributed to a better understanding of these processes, although the underlying regulatory mechanisms are still not fully elucidated and attempts to improve crop yield by modulating carbon partitioning were only partially successful. Plant pathogens also interfere with source–sink interaction. To this end they have evolved a wide range of sophisticated strategies to allow their systemic spread, suppression of plant defence and induction of sink function to support nutrient acquisition for their growth. Studying compatible interactions of plants and pathogens like viruses, bacteria and fungi can be exploited to investigate different levels of source–sink regulation. The identification of microbial factors and their host targets involved in regulation of plant primary metabolism may allow developing novel strategies to increase crop yield. Here we will discuss recent studies on plant–microbe interactions aimed at elucidating mechanisms of compatibility.     

Chemicobiological interactions and the use of partition coefficients in their correlation

Using octanol/water partition coefficients as an operational definition of hydrophobicity, 70 examples are given in which the hydrophobic interactions of organic compounds with themselves (in micelles) with macromolecules or with biological systems can be quantitatively correlated by the expression: log RBR = a log P + b. In this expression RBR is a binding constant or a relative biological response, P is the octanol/water partition coefficient and a and b are constants obtained via the method of least squares. These results are strong support for the utility of log P in the correlation of hydrophobic interactions. They also illustrate the extremely wide range of processes in which hydrophobic bonding plays a critical role.     

Molecular modelling of protein-carbohydrate interactions. Understanding the specificities of two legume lectins towards oligosaccharides

By means of a series of new molecular modelling tools, the conformationalbehaviour of mannose-containing di- and trisaccharides boundto either concanavalin A or Lathyrus ochrus isolectin I (LOLI)has been assessed. Tools for estimating and analysing eitherthe ‘rigid’ or the ‘relaxed’ potentialenergy surfaces, representing the conformational space availablefor carbohydrates once interacting with lectins, are reportedfor the first time. Restrictions of conformational space arepredicted to occur with different magnitudes, depending on thenature of the glycosidic linkages, as well as the size of thecarbohydrates. Results from these molecular modelling studiesare compared to existing structural data. Not only could theobserved conformations and orientations of carbohydrates incrystalline lectin–oligosaccharides complexes be reproduced,but several other likely situations were also predicted to occur.Entropy calculations have been performed for comparison withexperimental thermodynamics data. The results of the simulationcan also help giving an explanation of some observed affinityconstants at the molecular level. concanavalin A Lathyrus ochrus lectin-oligosaccharide molecular modelling     

Visualization of hydrogen atoms in a perdeuterated lectin-fucose complex reveals key details of protein-carbohydrate interactions

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Structural energetics of protein-carbohydrate interactions: Insights derived from the study of lysozyme binding to its natural saccharide inhibitors

High-sensitivity isothermal titration calorimetry was used to characterize the binding of the glycohydrolitic enzyme hen egg-white lysozyme to its natural saccharide inhibitors, chitobiose and chitrotriose. Measurements were done at a pH of 4.7, in the 15 degrees C -45 degrees C temperature range. Using a structural-energetic parameterization derived previously for lectin-carbohydrate associations, both binding enthalpies and entropies for the present systems and for the complex of chitobiose with turkey egg-white lysozyme from the literature were correctly accounted for. These observations suggest that both lysozymes and lectins follow the same structural-energetic behavior in the binding to their ligands. From the analysis of lysozyme data in conjunction with other binding data reported in the literature, an ad hoc parameterization of DeltaCp for protein-carbohydrate complexes was derived for the first time. The novel parameters for both polar and apolar surface areas differed significantly from correlations obtained previously from model compounds and protein-folding data. As DeltaCp is extremely sensitive to changes in solvent structure, this finding indicates that protein-carbohydrate complexes have distinctive hydration properties. According to our analysis, the dehydration of polar groups is the major cause for the observed decrease in DeltaCp, which implies that these groups behave hydrophobically. The contribution of apolar surface areas was found of the expected sign, but their specific weight is much smaller than those obtained in other correlations. This small contribution to DeltaCp is consistent with Lemieux's hypothesis of a low degree of hydration of apolar surfaces on carbohydrates.     

The use of fluoresceinphosphatidylethanolamine (FPE) as a real-time probe for peptide-membrane interactions

The characterization of fluorescelnphosphatidylethanolamlne (FPE) as a real-time Indicator of the electrostatic nature of a membrane surface is described. The conditions appropriate for the labelling of membranes and the implementation of FPE as a tool to monitor the interactions of various peptides with model membranes are outlined. It is shown that of the membrane-active peptides studied, Naja naja kaouthla cardiotoxin and pyrularia thionin bind to certain model membranes without insertion. Whereas the leader sequence of the nuclear encoded subunit IV of mammalian cytochrome c oxidase (E.C. 1.9.3.1), known as p-25, and melittin appear to bind and then partially insert into the membrane. It seems evident also that melittin does not adopt a fully transmembrane configuration. Melittin is known to promote membrane lysis and by employing a rapid-kinetic technique it is shown that the time-course of such lysis does not appear to correlate with peptide binding, but following binding a significant proportion of melittin must become inserted into the membrane before lysis appears to commence.