Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 1. Irreversible dissociation induced by sodium chloride concentration jumps

The dissociation kinetics of cooperatively bound bacteriophage T4 gene 32 protein from a variety of single-stranded homopolynucleotides has been investigated by stopped-flow techniques. Irreversible dissociation of the complexes was induced by rapidly increasing the salt concentration and monitoring the increase in tryptophan fluorescence upon dissociation of the gene 32 protein. The dependence of the apparent dissociation rate constant on initial fractional saturation of the nucleic acid lattice as well as the observation of zero-order kinetics when the lattice is initially fully saturated with protein indicates that dissociation occurs only from the ends of protein clusters and not from doubly contiguous molecules. The data for the entire time course are quantitatively fit by a kinetics model specifying irreversible dissociation of only singly contiguously bound protein [Lohman, T.M. (1983) Biopolymers 22, 1697-1713]. This model is used to extract molecular rate constants for the dissociation of isolated, singly contiguously and doubly contiguously bound protein. It is also shown that the polynucleotide specificity observed for the cooperative binding constant, K omega, and the cooperativity itself are intrinsic properties of the dissociation rate of the various complexes.     

Model for the irreversible dissociation kinetics of cooperatively bound protein–nucleic acid complexes

We present a quantitative model for the irreversible dissociation kinetics of cooperatively bound nonspecific protein–nucleic acid complexes. The model assumes that the major pathway of dissociation is via singly contiguously bound protein that “peels” off the ends of clusters of bound protein. It should therefore be most applicable for proteins that bind nucleic acids with high cooperativity (w > 10³). Furthermore, the model assumes that no redistribution of bound protein occurs during the time course of the dissociation. Solutions to the rate equations are presented for the entire time course of the dissociation. Under initial conditions such that the nucleic acid is less than fully saturated with protein, a single-exponential decay is predicted (if w is large). However, when the nucleic acid lattice is initially fully saturated, zero-order kinetics, corresponding to a constant rate of protein dissociation, is predicted. The experimental observation of zero-order dissociation kinetics in a cooperative protein–nucleic acid system is a good qualitative indicator for the dissociation mechanism discussed here. A discussion of the analysis of experimental data that enables one to extract molecular rate constants is presented. Furthermore, comparisons are made between the nonredistributing model presented here and Epstein's model [Epstein, I. R. (1979) Biopolymers 18 , 2037–2050] in which protein can translocate infinitely quickly while bound to the nucleic acid, and hence protein clusters redistribute during dissociation and maintain an equilibrium distribution on the nucleic acid at all times.     

Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 2. Changes in mechanism as a function of sodium chloride concentration and other solution variables

The dissociation kinetics of bacteriophage T4 coded gene 32 protein-single-stranded nucleic acid complexes have been examined as a function of monovalent salt concentration, temperature, and pH in order to investigate the details of the dissociation of cooperatively bound protein. Fluorescence stopped-flow techniques were used, and irreversible dissociation was induced by a combination of [NaCl] jumps and mixing with excess nucleic acid competitor. This made it possible to directly investigate the irreversible dissociation process over a wide range of NaCl concentrations [e.g., from 50 mM to 0.60 M for the gene 32 protein-poly(A) complex], in the absence of reassociation. Over the entire salt range, the only dissociable species observed is the singly contiguously bound gene 32 protein which dissociates from the ends of protein clusters. However, the [NaCl] dependence of the dissociation rate constant suggests that two competing pathways exist for dissociation of cooperatively bound gene 32 protein from the ends of protein clusters. At high monovalent salt concentrations, dissociation is dominated by a single-step process, with log ke/log [NaCl] = 6.5 +/- 0.5; i.e., the dissociation rate constant increases with increasing NaCl concentration due to the uptake of approximately six monovalent ions upon dissociation. This indicates that singly contiguous protein dissociates directly into solution. However, at much lower [NaCl] the data suggest that gene 32 protein, when bound at the end of a protein cluster, dissociates by first sliding off the end to form a noncooperatively bound intermediate which subsequently dissociates. A quantitative model which incorporates the sliding pathway [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948] in the dissociation mechanism fits the data reasonably well and suggests that noncooperatively bound monomers of gene 32 protein may be capable of one-dimensional translocation along single-stranded nucleic acids as suggested by independent kinetic data on the association reaction [Lohman, T. M., & Kowalczykowski, S. C. (1981) J. Mol. Biol. 152, 67-109]. It is also observed that both the absolute dissociation rate constant for T4 gene 32 protein and its salt dependence are sensitive to the average molecular weight and polydispersity of the nucleic acid sample used. This is a general phenomenon exhibited by proteins that bind to nucleic acids in a highly cooperative manner.     

New plot showing the existence of cooperativity, anticooperativity, and an arbitrary value of the neighbor-exclusion parameter in systems of ligand binding to linear biopolymers

A new way of plotting the isotherm for ligand binding to linear biopolymers is presented. In this plot the isotherm for noncooperative binding of ligands of length n (n-mers) becomes a straight line and the existence of cooperativity and anticooperativity between bound ligands is detected by appearance of opposite convexity of the curved isotherm. It is also usable for cases of fractional n values. Usefulness of the new plot in determining precise mechanisms of binding is shown using experimental data.     

Characterization of the interactions of an amino-terminal fibronectin fragment with the native molecule: Implications for polymerization of fibronectin

Plasma fibronectin is a 450-kD glycoprotein composed of two similar subunits connected by interchain disulfide bridges that may fold over in solution, allowing the amino terminus of each chain to bind the carboxyl terminus of the same subunit or a different subunit, thereby allowing polymerization. In order to study the characteristics of the fold-over interaction, the interaction between the amino terminal 29-kD fragment of fibronectin with native fibronectin has been studied in detail. One 29-kD molecule bound per fibronectin subunit, the apparent equilibrium dissociation constant was 40 nM, and the half-times for association and dissociation at 22°C were, respectively, 16 h and 23 days. Complexation could be inhibited by high concentrations of salt, but not by 8M urea. Amino terminal 20-kD and carboxyl terminal 8-kD subfragments of the 29-kD fragment also bound fibronectin and the activity was dependent on the integrity of the type 1 loop structures. The kinetics of the interaction of 29-kD fragment with fibronectin were unaffected by the presence of ligands, but were affected by detergents such as sodium dodecyl sulfate or deoxycholate, which enhanced the rate of interaction over 100-fold or 6-fold, respectively. Therefore, the interaction of fibronectin with ionic cell membrane components such as deoxycholate in vivo may trigger polymerization.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.     

Theoretical calculations of the helix–coil transition of DNA in the presence of large,cooperatively binding ligands

Theoretical calculations are conducted on the helix–coil transition of DNA, in the presence of large, cooperatively binding ligands modeled after the DNA-binding proteins of current biological interest. The ligands are allowed to bind both to helx and to coil, to cover up any number of bases or base pairs in the complex, and to interact cooperatively with their nearest neighbors. The DNA is treated in the infinite homogeneous Ising model approximation, and all calculations are done by Lifson's method of sequence-generating functions. DNA melting curves are calculated by computer in order to expolore the effects on the transition of ligand size, binding constant, free activity, and ligand–ligand cooperativity. The calculations indicate that (1) at the same intrinsic free energy change per base pair of the complexes, small ligands, for purely entropic reasons, are more effective than are large ligands in shifting the DNA melting temperature; (2) the response of the DNA melting temperature to increased ligand binding constant K and/or free ligand activity L is adequately represented at high values of KL (but not at low KL) by a simple independent site model; (3) if curves are calculated with the total amount of added ligand remaining constant and the free ligand activity allowed to vary throughout the transition, biphasic melting curves can be obtained in the complete absence of ligand–ligand cooperativity. In an Appendix, the denaturation of poly[d(A-T)] in the presence of the drug, netropsin, is used to verify some features of the theory and to illustrate how the theory can be used to obtain numerical estimates of the ligand binding parameters from the experimental melting curves.     

Ionic strength perturbation kinetics of gene 32 protein dissociation from its complex with single-stranded DNA.

Equilibrium and kinetic studies of the interaction of gene 32 protein of T4 phage with single-stranded fd DNA were performed monitoring the changes in protein fluorescence. From the fluorescence titrations, it was estimated that a monomer of gene 32 protein covered six nucleotide bases on the DNA and the lower limit for the apparent association constant was 1.9 x 10(8) M-1 with a cooperative parameter of 10(3) in 0.1 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride (pH 7) at 25 degrees C. When an ionic strength jump was applied to the gene 32 protein-fd DNA complex using a stopped-flow apparatus, the complex underwent a dissociation into its individual components accompanied by an increase in protein fluorescence. The kinetics of the dissociation are not consistent with a single first-order process. The data, however, can be analyzed in terms of a model in which gene 32 protein molecules release cooperatively starting from either one or both ends of a cluster of proteins bound to fd DNA. This type of dissociation of gene 32 protein from single-stranded DNA is very efficient and has interesting implications: it could provide a way to facilitate a rapid "zippering" of the two complementary DNA strands during DNA replication and genetic recombination.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide

Due to the lack of an outer membrane, Gram-positive bacteria (e.g., Bacillus species) are considered as promising host organisms for the secretory production of biotechnologically relevant heterologous proteins. However, the yields of the desired target proteins were often reported to be disappointingly low. Here, we used saturation mutagenesis of the positively charged N-domain (positions 2–7) of the signal peptide of the Bacillus subtilis α-amylase (AmyE) as a novel approach for the improvement of the secretion of a heterologous model protein, cutinase from Fusarium solani pisi, by the general secretory pathway of B. subtilis. Automated high-throughput screening of the resulting signal peptide libraries allowed for the identification of four single point mutations that resulted in significantly increased cutinase amounts, three of which surprisingly reduced the net charge of the N-domain from +3 to +2. Characterization of the effects of the identified mutations on protein synthesis and export kinetics by pulse-chase analyses indicates that an optimal balance between biosynthesis and the flow of the target protein through all stages of the B. subtilis secretion pathway is of crucial importance with respect to yield and quality of secreted heterologous proteins.     

Monte carlo simulations of protein assembly, disassembly, and linear motion on DNA

We use Monte Carlo simulations to analyze the simultaneous interactions of multiple proteins to a long DNA molecule. We study the time dependence of protein organization on DNA for different regimes that comprise (non)cooperative sequence-independent protein assembly, dissociation, and linear motion. A range of different behaviors is observed for the dynamics, final coverage, and cluster size distributions. We observe that the DNA substrate is almost never completely covered by protein when taking into account only (non)cooperative binding, because gaps remain on the substrate that are smaller than the binding site size of the protein. Due to these gaps, the apparent binding size of a protein during noncooperative binding can be overestimated by up to 30%. During dissociation of cooperatively bound proteins, the dissociation curve can be exponentially shaped even when allowing only end-dependent dissociation. We discuss the potential of our method for the analysis of a number of single-molecule experiments, for example, the binding of the DNA-repair proteins RecA and Rad51 to DNA.     

Interplay between binding affinity and kinetics in protein–protein interactions

To clarify the interplay between the binding affinity and kinetics of protein–protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound‐state valley is deep with a barrier height of 12 ? 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920–933. © 2016 Wiley Periodicals, Inc.     

Equilibrium dialysis studies of polyamine binding to DNA

Binding constants and binding site sizes for the interactions of the polyamines spermine (+4), spermidine (+3), and putrecine (+2) with helical DNA have been determined as a function of ionic conditions and temperature by equilibrium dialysis using ¹⁴C-labeled polyamines. In addition, competition equilibrium dialysis has been used to determine binding parameters for the divalent cations putrescine and Mg²⁺ from the competitive effect of these ions on the binding of spermine or spermidine. In all cases, the logarithm of the binding constant (log K_obs) varies linearly with the logarithm of the monovalent salt concentration; the slopes d log K_obs/d log[NaCl] are proportional to the valence of the ligand, and values of the extrapolated binding constants at 1M NaCl obtained from the intercepts are small (of order 1–10M^?1). In those cases examined, K_obs is insensitive to temperature; the free energy of binding is predominantly entropic. Consequently, polymines as DNA-binding ligands behave analogously to the oligolysìnes investigated previously [cf. Record, Lohman & de Haseth (1976) J. Mol. Biol. 107 , 145–158; Lohman, de Haseth & Record (1980) Biochemistry 19 , 3522–3530]. The interactions of these oligocations with DNA are predominantly electrostatic and are driven by the release of thermodynamically bound electrolyte ions from the vicinity of the DNA. The extent to which these oligocations are localized at individual phosphate binding sites or delocalized on the DNA molecule is currently not known.     

DNA-binding properties of gene-5 protein encoded by bacteriophage M 13. 1. The kinetics of the dissociation of gene-5-protein.polynucleotide complexes upon addition of salt

The irreversible dissociation kinetics of complexes of M13-encoded gene-5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped-flow experiments. A linear decay was found for all gene-5-protein.poly(dA) complexes and for the gene-5-protein.M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single-exponential decay was found for the dissociation of gene-5-protein.M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel is not maintained. The binding of gene-5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA-binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide. The absence of line broadening for gene-5-protein.polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.     

Studies on interaction of oligopeptides with sodium dodecyl sulfate: Stopped-flow kinetics of chemical modification of tryptophan residues withN-bromosuccinimide

The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) _n .     

The dependence on ammonia pretreatment of N−O activation by Co(II) sites in zeolites: a DFT and ab initio molecular dynamics study

This work is focused on the donor properties of cobalt-exchanged cationic sites in zeolites. It is based on cluster and periodic density functional theory modeling for relevant {[Co(II)(NH₃)_n]–NO} adducts, where Co(II) means a cobalt cation embedded either in a periodic model of chabasite (CHA) zeolite or in model clusters. NO stretching frequencies were derived from MD trajectories and compared to harmonic values from cluster calculations. By relating calculated NO frequencies to experimental FTIR spectra, it was shown that the forms of {Co(II)-NO} adducts comprising three or four ammonia co-ligands dominate the spectrum taken in ammonia-saturation conditions while forms with two NH₃ ligands prevail under intermediate ammonia saturation. Finally, this work confirms the critical dependence of Co(II) activation ability towards NO upon the center donor properties, reinforced by ligation of strong donor ammonia ligands. However, strongly bound ligands appear also to compete with interaction of the center with the electron-rich framework, and a balance must be observed to maintain optimal activation ability.

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Graphical abstract A snapshot from MD trajectory showing a fragment of periodic framework with twoCo(II)–NO centers, bound to one framework oxygen and strongly coordinating three ammonia ligands with four others forming the second coordination sphere

     

[125I]RTI-55 Binding to Cocaine-Sensitive Dopaminergic and Serotonergic Uptake Sites in the Human Brain

[¹²⁵I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [¹²⁵I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [¹²⁵I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [¹²⁵I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with K_D= 66 ± 35 pM and S_max= 13.2 ± 10.1 pmol/g of tissue and a low-affinity site with K_D= 1.52 ± 0.55 nM and B_max of 47.5 ± 11-2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 > GBR-12909 > mazindol > WIN 35428 > = methylphenidate > (?)-cocaine > buproprion > (±)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with K_D= 370 ± 84 pM. It appears that [¹²⁵I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.