Cooperative ligand-lattice binding. Approximate Gaussian binding distribution

Nearest-neighbor cooperative binding of a ligand covering n sites and binding with equilibrium constant K and cooperativity factor omega to a large molecule with m binding sites (m much greater than n omega, n/omega) can be approximately described by a Gaussian distribution P(q-qmax), where q is the number of ligands bound and qmax the most probable value of q. The variance of the Gaussian is equal to the derivative dqmax/d ln(L), where L is the free ligand concentration. This variance, sigma 2, is a complicated function of qmax. However, in the limits of very large cooperativity, omega much greater than 1, very large anticooperativity, omega much less than 1, or noncooperativity, omega = 1, simpler expressions for sigma 2 can be given. For qmax = m/(n + 1), where the most probable number of bound ligands equals the number of free binding sites, sigma 2 has a particularly simple form: sigma 2 = 2m omega 1/2/(n + 1)3. The Gaussian and the infinite lattice approximations for the average number of ligands bound are good approximations only if sigma is much smaller than the number of binding sites. The variance may therefore provide an easy check on the validity of the infinite lattice approximation, which is commonly used to analyze experimental binding data.     

Laminin binding proteins.

Cells express many proteins that bind to laminin, the major adhesive component of basement membranes. Some of these, specifically integrins, function as transmembrane receptors that 'signal' the presence of laminin on the cell surface to the cytoplasm. Lectins constitute a second class of laminin binding proteins that may augment integrin function by interacting with laminin carbohydrate. Caution must be used in ascribing functions to other laminin binding proteins, especially cytosolic proteins.     

Specific binding of T lymphocytes to macrophages. I. Kinetics of binding.

Peritoneal exudate lymphocytes obtained from immune guinea pigs and cultured for 1 week on antigen-pulsed autologous macrophages were tested for their ability to bind to fresh antigen-pulsed autologous macrophages or to macrophages pulsed with an irrelevant antigen. Up to 30% of the lymphocytes bound to macrophages bearing the relevant antigen whereas only 2 to 5% remained nonspecifically bound to macrophages after vigorous washing. Specific binding was observed in cultures as early as 1 hr. Analysis of the kinetics of binding suggests that the observed nonspecific binding is not a step in specific binding. The possibility that weaker antigen-independent association between lymphocytes and macrophages precedes specific binding cannot be excluded. No evidence was obtained that serum antibody adsorbed to the macrophage or T cell plays a role in this cell interaction or that the T cell can bind antigen directly. We suggest that the observed specific binding represents the initial event in stimulation of T lymphocytes by antigen.     

Ligand binding to proteins: the binding landscape model.

Models of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand-induced denaturation, ANS and heme-like binding, and "lock-and-key" and "induced-fit" specific binding behaviors characterized by Michaelis-Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis-Menten isotherms. Even low-population binding to denatured states can cause changes in global stability, hydrogen-exchange rates, and thermal B-factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass-action models.     

Cooperative binding of polyamines induces the Escherichia coli single-strand binding protein-DNA binding mode transitions.

The Escherichia coli single-strand binding (SSB) protein is an essential protein involved in DNA replication, recombination, and repair processes. The tetrameric protein binds to ss nucleic acids in a number of different binding modes in vitro. These modes differ in the number of nucleotides occluded per SSB tetramer and in the type and degree of cooperative complexes that are formed with ss DNA. Although it is not yet known whether only one or all of these modes function in vivo, based on the dramatically different properties of the SSB tetramer in these different ss DNA binding modes, it has been suggested that the different modes may function selectively in replication, recombination, and/or repair. The transitions between these different modes are very sensitive to solution conditions, including salt (concentration, as well as cation and anion type), pH, and temperature. We have examined the effects of multivalent cations, principally the polyamine spermine, on the SSB-ss poly(dT) binding mode transitions and find that the transition from the (SSB)35 to the (SSB)56 binding mode can be induced by micromolar concentrations of polyamines as well as the inorganic cation Co(NH3)6(3+). Furthermore, these multivalent cations, as well as Mg2+, induce the binding mode transition by binding cooperatively to the SSB-poly(dT) complexes. These observations are interesting in light of the fact that polyamines, such as spermidine, are part of the ionic environment in E. coli and hence these cations are likely to affect the distribution of SSB-ss DNA binding modes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor binding of asialoerythropoietin.

The interaction of 125I-asialoerythropoietin (asialoepo) with receptors has been characterized both by binding assay and affinity cross-linking. Purified spleen cells from mice infected with the anemia strain of Friend virus (FVA cells) have receptors for 125I-asialoepo with two classes of affinity constant: one with Kd = 0.02-0.03 nM and 300-400 per cell, the other with lower affinity (Kd = 0.9-1.2 nM) and 1,000-1,200 per cell. The Kd value for the high affinity site is one-third of that for the binding of native 125I-erythropoietin (125I-epo) to the same FVA cells (Kd = 0.08-0.1 nM). Using 125I-asialoepo or 125I-epo affinity cross-linking methods, we find two components with apparent molecular weights of 88 kDa and 105 kDa in FVA cells, and in the transformed mouse cell lines, 201, IW32, and NN10, in agreement with earlier studies using 125I-epo. These results indicate that 125I-asialoepo binds to the same receptors as 125I-epo, but with greater affinity for the high affinity site. Since 201 cells contain only a single class of lower affinity receptors for erythropoietin (epo), finding the same two components as found for FVA cells by cross-linking experiment indicates that the two components do not represent the two classes of receptor.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site.

As model compounds for Ni(II)-binding heparin-like compounds isolated from human kidneys (Templeton, D.M. & Sarkar, B. (1985) Biochem. J. 230 35-42.), we investigated two disaccharides--4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-2,5-anhydro- D-mannitol, disodium salt (1a), and 4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-6-O- sulfo-2,5-anhydro-D-mannitol, trisodium salt (1b)--that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate (1a) was active whereas the disulfate (1b) was inactive in a high-performance liquid chromatography (HPLC) binding assay with the tracer ions 63Ni(II) 54Mn(II), 65Zn(II), and 109Cd(II). This result is in accord with the isolation of two 67Cu(II) and 63Ni(II) binding fractions from a complete pool of nitrous-acid-derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound (1a) and the other as a tetrasulfated trisaccharide by high resolution FAB-MS, NMR and HPLC-PAD. Similarly, two synthetic disaccharides-methyl, 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-alpha-D-glucosamine, trisodium salt [IdopA2S(alpha 1,4)GlcNS alpha Me, 2a], and 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-6-O-sulfo- alpha-D-glucosamine, tetrasodium salt [IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b]--were shown to bind tracer amounts of 63Ni and 67Cu using chromatographic assays. Subsequently, 1H NMR titrations of 1a, 1b, 2a, and 2b with Zn (OAc)2 were analyzed to yield 1:1 Zn(II)-binding constants of 472 +/- 59, 698 +/- 120, 8,758 +/- 2,237 and 20,100 +/- 5,598 M-1, respectively. The values for 2a and 2b suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 1H and 13C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions.     

Conformational stability and binding properties of porcine odorant binding protein.

Apparently homogeneous odorant binding protein purified from pig nasal mucosa (pOBP) exhibited subunit molecular masses of 17 223, 17 447, and 17 689 (major component) Da as estimated by ESI/MS. According to gel filtration, this protein, its truncated forms, and/or its variants are homodimeric under physiologic conditions (pH 6-7, 0.1 M NaCl). The dimer if monomer equilibrium shifts toward a prevalent monomeric form at pH <4.5. Velocity sedimentation reveals a monomeric state of OBP at both pH 7.2 and 3.5, indicating a pressure-induced dissociation of the homodimer. High-sensitivity differential scanning calorimetry (HS-DSC) shows that the unfolding transition of pOBP is reversible at neutral pH. It is characterized by the transition temperature of 69.23 degrees C and an enthalpy of 391.1 kJ/mol per monomer. The transition heat capacity curve of pOBP is well-approximated by the two-state model on the level of subunit, indicating that the two monomers behave independently. Isothermal titration calorimetry (ITC) shows that at physiological pH pOBP binds 2-isobutyl-3-methoxypyrazine (IBMP) and 3,7-dimethyloctan-1-ol (DMO) with association constants of 3.19 x 10(6) and 4.94 x 10(6) M(-)(1) and enthalpies of -97.2 and -87.8 kJ/mol, respectively. The binding stoichiometry of both ligands is nearly one molecule of ligand per homodimer of pOBP. The interaction of pOBP with both ligands is enthalpically driven with an unfavorable change of entropy. The binding affinity of pOBP with IBMP does not change significantly at acidic pH, while the binding stoichiometry is nearly halved. According to HS-DSC data, the interaction with IBMP and DMO leads to a substantial stabilization of the pOBP folded structure, which is manifested by the increase in the unfolding temperature and enthalpy. The calorimetric data allow us to conclude that the mechanism of binding of the studied odorants to pOBP is not dominated by a hydrophobic effect related to any change in the hydration state of protein and ligand groups but, most likely, is driven by polar and van der Waals interactions.     

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.     

Stem cell factor binding to retrovirus primer binding site silencers.

Using modified nuclear lysis and binding conditions, we have examined the binding of an embryonal carcinoma (EC) cell factor, binding factor A, to a stem cell-specific silencer which acts at the DNA level and overlaps the Moloney murine leukemia virus (M-MuLV) proline primer binding site (PBS). Following our protocol, we found that in vitro binding of factor A correlated with the in vivo activity of the M-MuLV silencer. Factor A bound specifically to the wild-type silencer element at room temperature and 30 degrees C, but not at 4 degrees C, and bound 10-fold better to the full-length silencer than to a minimal silencer core element. The factor was enriched in nuclear compared with cytosolic extracts and in undifferentiated EC cells compared with differentiated cells in which the silencer is nonfunctional. Salt and ion requirements for factor A binding were investigated, and partial purification steps indicated the factor to be a heparin-Sepharose-binding moiety of greater than 100 kDa. To examine possible relationships between silencer and PBS activities, sequences representing phenylalanine, isoleucine, lysine-1,2, lysine-3, methionine, and tryptophan PBS DNA fragments were tested in vivo for stem cell-specific repression of M-MuLV expression and in vitro in DNA binding assays. Of these PBS elements, only the lysine-1,2 PBS DNA fragment showed consistently high levels of repression. Interestingly, the lysine-1,2 PBS DNA fragment also formed a complex with an EC cell factor with characteristics similar to those of factor A. However, the two factors did not cross-compete in binding studies, suggesting that they may be different but related factors. Our results suggest that expression of Mason-Pfizer monkey virus, visna virus, and spumavirus, which use the lysine-1,2 PBS, may be inhibited in undifferentiated stem cells.     

Stabilization of the FK506 binding protein by ligand binding.

Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed.     

Studies of antigen binding on human basophils. I. Antigen binding and functional consequences

We developed an assay to study simultaneously antigen binding and its functional consequences on human basophils. Using a 125iodine-labeled penicillin-human serum albumin conjugate, we were able to detect as few as 100 molecules of antigen bound to purified basophils. We found that histamine release could be initiated with fewer than 100 molecules of antigen and that the optimum for histamine release occurred at a concentration at which 10 to 15% of the available antibody-binding sites were occupied. Analysis of the binding kinetics in the presence of monovalent hapten allowed a calculation of the affinity constant for the antibody:hapten association; the value of 2 to 3 X 10(6)/Msec confirmed an earlier independent calculation. Binding data also suggested a 25% fraction of the bound antigen was binding in monogamous bivalent form. It is anticipated that studies of this kind will delineate the nature of the antibody-antigen reaction on cell surfaces.     

Spin assay as a general method for studying plasma protein binding. Bilirubin-albumin binding.

A mutant of the Chinese Hamster Ovary cell line, CHO-K1, has been isolated which is resistant to killing by 25-hydroxy cholesterol in the absence of cholesterol. There is no effect on acetate incorporation into cholesterol by 25-hydroxy cholesterol in the mutant under conditions in which incorporation is inhibited in the parent cell. The mutant also appears to be defective in the regulation of cholesterol synthesis by serum choleserol.     

Specific and cooperative binding of E. coli single-stranded DNA binding protein to mRNA.

Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.     

Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.     

Conservation of binding site specificity of three yeast DNA binding proteins.

Sequence specific binding of protein extracts from 13 different yeast species to three oligonucleotide probes and two points mutants derived from Saccharomyces cerevisiae DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites for GRF1/RAP1/ABF1 and CP1/CPF1. Most yeasts in the genus Saccharomyces showed specific binding to all three probes and also displayed similar sequence requirements when challenged by molar excesses of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPF1 binding activity was the most widespread, while the other two were more limited.