A simple mathematical model of cooperativity in receptor mosaics based on the "symmetry rule"

The phenomenon of receptor-receptor interactions was hypothesized about 20 years ago. It has been demonstrated by now that receptor-receptor interactions between G-protein coupled receptors (GPCRs) occur at plasma membrane level and result in the reciprocal modulation of their binding characteristics (i.e., cooperativity). One of the most important feature of this phenomenon is the concept of cluster of receptors, or receptor mosaic (RM). However, no proper mathematical approach has still been available to characterize RMs as far as their receptor composition, receptor topography and order of receptor activation inside the RM. This paper tries to fill the gap. A simple mathematical approach to the cooperativity in RMs formed by dimers of identical receptors and/or by iso-receptors is proposed. To this aim the so-called "symmetry rule" has been considered. This approach allows to describe by means of a simple energy function the effects of receptor composition (number of dimers), spatial organisation (respective location of the dimers) and order of activation (order according to which the single receptors are ligated) on the integrative cooperativity (index) of the RMs.     

Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis

We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA(2)B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell's genome. Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.     

Multiple Escherichia coli RecQ Helicase Monomers Cooperate to Unwind Long DNA Substrates: A FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY STUDY*

The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3′-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 °C, whereas only modest or no cooperativity was observed at 25 °C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 °C but not at 25 °C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.     

The insulin receptor: a prototype for dimeric, allosteric membrane receptors?

The recent crystallographic structure of the insulin receptor (IR) extracellular domain has brought us closer to ending several decades of speculation regarding the stoichiometry and mechanism of insulin-receptor binding and negative cooperativity. It supports a bivalent crosslinking model whereby two sites on the insulin molecule alternately crosslink two partial-binding sites on each insulin-receptor half. Ligand-induced or -stabilized receptor dimerization or oligomerization is a general feature of receptor tyrosine kinases (RTKs), in addition to cytokine receptors, but the kinetic consequences of this mechanism have been less well studied in other RTKs than in the IR. Surprisingly, recent studies indicate that constitutive dimerization and negative cooperativity are also ubiquitous properties of G-protein-coupled receptors (GPCRs), which show allosteric mechanisms similar to those described for the IR.     

Biological physics in México

Biological and physical sciences possess a long-standing tradition of cooperativity as separate but related subfields of science. For some time, this cooperativity has been limited by their obvious differences in methods and views. Biological physics has recently experienced a kind of revival (or better a rebirth) due to the growth of molecular research on animate matter. New avenues for research have been opened for both theoretical and experimental physicists. Nevertheless, in order to better travel for such paths, the contemporary biological physicist should be armed with a set of specialized tools and methods but also with a new attitude toward multidisciplinarity. In this review article, we intend to somehow summarize what has been done in the past (in particular, as an example we will take a closer look at the Mexican case), to show some examples of fruitful investigations in the biological physics area and also to set a proposal of new curricula for physics students and professionals interested in applying their science to get a better understanding of the physical basis of biological function.     

Ca++ binding properties of human prothrombin.

The binding of Ca++ to human prothrombin has been investigated by equilibrium dialysis. The protein exhibited a positive cooperativity phenomenon for the first three Ca++ bound. Eleven to twelve Ca++ binding sites have been found. They could be differentiated in terms of two classes of sites with respect to their Ca++ affinity: 5 strong binding sites (log Kassoc = 3.9) and 7 weak binding sites (log Kassoc = 2.9). We attempted to determine the Hill coefficient of the strong binding sites responsible for cooperativity. Results have been compared to data previously reported for bovine prothrombin.     

Allosteric regulation, cooperativity, and biochemical oscillations

Allosteric regulation is associated with a number of periodic phenomena in biochemical systems. The cooperative nature of such regulatory interactions provides a source of nonlinearity that favors oscillatory behavior. We assess the role of cooperativity in the onset of biochemical oscillations by analyzing two specific examples. First, we consider a model for a product-activated allosteric enzyme which has previously been proposed to account for glycolytic oscillations. While enzyme cooperativity plays an important role in the occurrence of oscillations, we show that these may nevertheless occur in the absence of cooperativity when the reaction product is removed in a Michaelian rather than linear manner. The second model considered was recently proposed to account for signal-induced oscillations of intracellular calcium. This phenomenon originates from a nonlinear process of calcium-induced calcium release. Here also, the cooperative nature of that positive feedback favors the occurrence of oscillations but is not absolutely required for periodic behavior. Besides underlining the importance of cooperativity, the results highlight the role of diffuse nonlinearities distributed over several steps within a regulated system: even in the absence of cooperativity, such mild nonlinearities (e.g., of the Michaelian type) may combine to raise the overall degree of nonlinearity up to the level required for oscillations.     

Urea effect on Cu(2+)-induced DNA structural transitions in solution

Cu(2+) ion interaction with DNA in aqueous solutions containing urea (0-5 M) was studied by IR spectroscopy. It was shown that upon the Cu(2+) ion binding DNA transition into a compact form occurs. This transition is of positive cooperativity. We suppose that the mechanism of Cu(2+)-induced DNA compaction in solutions containing urea is not completely electrostatic. Urea addition to the DNA solution decreases the Cu(2+) ion concentration required to induce DNA compaction. As the urea content in solution rises, the binding constant of Cu(2+) ions interacting with DNA increases, going through the maximum in the case of 2 M solution; further increase of the urea content in solutions leads to decrease of the binding constant. DNA transition into the compact form under the Cu(2+) ion action is determined not only by the effects of the solution dielectric permeability but by the solvation effects; when changes of the dielectric permeability are small the solvation effects may prevail. Urea addition to the DNA solution also decreases cooperativity of the DNA compaction process. Perhaps, cooperativity of the DNA transition into the compact state depends on the ordered spatial structure of water adjacent to the macromolecule and decreases on the structure destruction.     

Intrauterine growth restriction leads to changes in sulfur amino acid metabolism, but not global DNA methylation, in Yucatan miniature piglets

Intrauterine growth restriction (IUGR), in both animals and humans, has been linked to metabolic syndrome later in life. There has been recent evidence that perturbations in sulfur amino acid metabolism may be involved in this early programming phenomenon. Methionine is the precursor for cellular methylation reactions and for the synthesis of cysteine. It has been suggested that the mechanism behind the "fetal origins" of adult diseases may be epigenetic, involving DNA methylation. Because we have recently demonstrated the fetal origins phenomenon in Yucatan miniature swine, we hypothesized that sulfur amino acid metabolism is altered in IUGR piglets. In this study, metabolites and the activities of sulfur amino acid cycle enzymes were analyzed in liver samples of 3- to 5-day-old runt (IUGR: 0.85±0.13 kg) and large (1.36±0.21 kg) Yucatan miniature pig littermates (n=6 pairs). The IUGR piglets had significantly lower specific and total activities of betaine-homocysteine methyltransferase (BHMT) and cystathionine γ-lyase (CGL) than larger littermates (P<.05). Expression of CGL (but not BHMT) mRNA was also lower in IUGR piglets (P<.05). This low CGL reduced cysteine and taurine concentrations in IUGR pigs and led to an accumulation of hepatic cystathionine, with lower homocysteine concentrations. Methylation index and liver global DNA methylation were unaltered. Reduced prenatal growth in Yucatan miniature piglets impairs their remethylation capacity as well as their ability to remove cystathionine and synthesize cysteine and taurine, which could have important implications on long-term health outcomes of IUGR neonates.     

Apoptotic DNase network: Mutual induction and cooperation among apoptotic endonucleases

DNA fragmentation produced by apoptotic DNases (endonucleases) leads to irreversible cell death. Although apoptotic DNases are simultaneously induced following toxic/oxidative cell injury and/or failed DNA repair, the study of DNases in apoptosis has generally been reductionist in approach, focusing on individual DNases rather than their possible cooperativity. Coordinated induction of DNases would require a mechanism of communication; however, mutual DNase induction or activation of DNases by enzymatic or non-enzymatic mechanisms is not currently recognized. The evidence presented in this review suggests apoptotic DNases operate in a network in which members induce each other through the DNA breaks they produce. With DNA breaks being a common communicator among DNases, it would be logical to propose that DNA breaks from other sources such as oxidative DNA damage or actions of DNA repair endonucleases and DNA topoisomerases may also serve as triggers for a cooperative DNase feedback loop leading to elevated DNA fragmentation and subsequent cell death. Therefore, mutual induction of apoptotic DNases has serious implications for studies focused on activation or inhibition of specific DNases as a strategy for therapeutic intervention aimed at modulation of cell death.     

Site-directed mutagenesis of a tetrameric dandelion polyphenol oxidase (PPO-6) reveals the site of subunit interaction

Polyphenol oxidases (PPOs) catalyze the oxidation of ortho-diphenols to the corresponding quinones (EC 1.10.3.1). In plants PPOs appear in gene families, and the corresponding isoenzymes are located to the thylakoid lumen of chloroplasts. Although plant PPOs are often discussed with regard to their role in defense reactions, a common physiological function has not yet been defined. We analyzed a tetrameric PPO isoenzyme (PPO-6) from dandelion (Taraxacum officinale) heterologously expressed in Escherichia coli, and found it to display cooperativity in catalysis, a phenomenon that has rarely been shown for plant PPOs previously. The identification of a surface-exposed cysteine (197) through molecular modeling followed by site-directed mutagenesis proved this amino acid residue to stabilize the tetramer via a disulfide linkage. The C197S-mutein still forms a tetrameric structure but shows impaired enzymatic efficiency and cooperativity and a reduction in stability. These findings indicate that oligomerization may be a physiological requirement for PPO-6 stability and function in vivo and raise new questions regarding distinct functions for specific PPO isoenzymes in plants.     

Self-assembly of double-stranded DNA molecules at nanomolar concentrations

Some proteins have the property of self-assembly, known to be an important mechanism in constructing supramolecular architectures for cellular functions. However, as yet, the ability of double-stranded (ds) DNA molecules to self-assemble has not been established. Here we report that dsDNA molecules also have a property of self-assembly in aqueous solutions containing physiological concentrations of Mg2+. We show that DNA molecules preferentially interact with molecules with an identical sequence and length even in a solution composed of heterogeneous DNA species. Curved DNA and DNA with an unusual conformation and property also exhibit this phenomenon, indicating that it is not specific to usual B-form DNA. Atomic force microscopy (AFM) directly reveals the assembled DNA molecules formed at concentrations of 10 nM but rarely at 1 nM. The self-assembly is concentration-dependent. We suggest that the attractive force causing DNA self-assembly may function in biological processes such as folding of repetitive DNA, recombination between homologous sequences, and synapsis in meiosis.     

Cooperativity-based modeling of heterotypic DNA nanostructure assembly

DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction—in which arms are unequal—as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations.     

Physical studies of the interaction between the Escherichia coli DNA binding protein and nucleic acids.

The interaction of nucleic acid with the Escherichia coli DNA-binding protein has been studied by fluorescence emission spectroscopy and sedimentation velocity analysis. The protein binds to single-strand DNA with an apparent equilibrium dissociation constant of 2 X 10(-9). It binds to the homopolymers poly (dA) and poly (dT) slightly more tightly, but has a larger apparent equilibrium dissociation constant to poly (dC). The protein also binds tightly to ribohomopolymers and to tRNA, but not to duplex DNA. By the use of defined-length oligonucleotides, it has been shown that the protein binds to DNA in a highly cooperative manner. The extent of cooperativity is seen as the difference in binding between an isolated monomeric protein molecule bound to DNA and two or more molecules binding to contiguous sites.     

DNA photolyases: physical properties, action mechanism, and roles in dark repair

DNA photolyases catalyze the light-dependent repair of cis,syn-cyclobutane dipyrimidines (pyrimidine dimers). Although the phenomenon of enzymatic photoreactivation was first described 40 years ago and photolyases were the first enzymes shown unequivocally to effect DNA repair, it has only been in the last 8 years that sufficient quantities of the enzymes have been purified to permit detailed studies of their physical properties, identification of their intrinsic chromophores, and elucidation of the mechanisms of dimer recognition and photolysis. In addition several of the genes encoding these enzymes have now been cloned and sequenced. These studies have revealed remarkable functional and structural conservation among these evolutionarily ancient enzymes and have identified a new role for photolyases in dark-repair processes which has implications for the mechanism of nucleotide excision repair in both prokaryotes and eukaryotes.     

Analysis of binding interactions between histone core complex and simian virus 40 DNA. A comparison of acetylated versus non-acetylated histone core complexes

The interactions of acetylated and non-acetylated core complex histones with simian virus 40 (SV40) DNA 1 have been analyzed. A modified filter-binding assay utilizing micrococcal nuclease, which allows quantification of histone octamer binding to DNA has been developed. Using this assay it was determined that both non-acetylated core complex histones ad core complex histones acetylated with acetyl adenylate to levels existing in vivo bind cooperatively to SV40 DNA 1. Although both interactions are cooperative, the magnitude of the cooperativity parameter, omega, is significantly less in the acetylated case. This difference in cooperativity is in contrast to the nearly identical intrinsic association constant, K, observed in both cases.     

Studying compaction-decompaction of DNA molecules induced by surfactants

The mechanism and detailed processes of DNA compaction and decompaction are essential for the life activities, as well as for the researches in the molecular biology, genetics and biomedicine. The compaction of two kinds of DNA molecules caused by Cetyltrimethyl Ammonium Bromide (CTAB) and their decompaction induced with sodium dodecyl sulfate (SDS) or excessive amount of CTAB have been investigated with multiple perspectives such as the UV-VIS spectrophotometry, dynamic light scattering, and zeta potential. The compaction phenomenon of DNA can easily be observed when the CTAB combines with the DNA, not just when the molar ratio Q_CTAB/Q_DNA is approximately equal to 1 as the conventional recognition, but also when Q_CTAB/Q_DNA <1,DNA can be compacted; Molecular state of DNA is only changed in the conformational structure, but not in the chemical structure. Finally, a model is suggested to help catch on the biophysical mechanism of DNA chain conformational change.     

Isolating Contributions from Intersegmental Transfer to DNA Searching by Alkyladenine DNA Glycosylase

Large genomes pose a challenge to DNA repair pathways because rare sites of damage must be efficiently located from among a vast excess of undamaged sites. Human alkyladenine DNA glycosylase (AAG) employs nonspecific DNA binding interactions and facilitated diffusion to conduct a highly redundant search of adjacent sites. This ensures that every site is searched, but could be a detriment if the protein is trapped in a local segment of DNA. Intersegmental transfer between DNA segments that are transiently in close proximity provides an elegant solution that balances global and local searching processes. It has been difficult to detect intersegmental transfer experimentally; therefore, we developed biochemical assays that allowed us to observe and measure the rates of intersegmental transfer by AAG. AAG has a flexible amino terminus that tunes its affinity for nonspecific DNA, but we find that it is not required for intersegmental transfer. As AAG has only a single DNA binding site, this argues against the bridging model for intersegmental transfer. The rates of intersegmental transfer are strongly dependent on the salt concentration, supporting a jumping mechanism that involves microscopic dissociation and capture by a proximal DNA site. As many DNA-binding proteins have only a single binding site, jumping may be a common mechanism for intersegmental transfer.     

Cooperative binding of m-AMSA to nucleic acids.

The equilibrium binding of the antitumor agent m-AMSA (4'-(9-acridinylamino) methane-sulfon-m-ansidide) has been examined by optical methods. These studies which have focused on the low bound drug concentrations (r values less than 0.02, base pairs) reveal m-AMSA to bind calf thymus DNA in a highly cooperative manner as indicated by the initial positive slope of the Scatchard plot. In contrast, the studies on the parent 9-aminoacridine under identical conditions demonstrate that this compound binds DNA in a noncooperative (neighbor exclusion) manner. The positive cooperative binding phenomenon of m-AMSA is probed as a function of ionic concentration and shown to exist over the range of salt concentrations examined (0.01 to 0.1 M); however, the magnitude of the cooperative binding is altered. This observation of cooperativity is consistent with earlier studies on biologically active compounds and may be related to such binding parameters as binding sequence selectivity and/or structural perturbations to the DNA structure.