New approaches for the analysis of molecular recognition using the IAsys evanescent wave biosensor

Trends in the analysis of molecular recognition using the IAsys evanescent wave biosensor are outlined. Diversification of sensor surface chemistry, an open cuvette format and the advent of robotics controlled by intelligent software are widening the range and throughput of applications. Analyses of binding and dissociation are now carried out across a wide spectrum of biomolecules, including protein, nucleic acid, carbohydrate and lipid. Determinations are obtained from a range of experimental formats. These include qualitative 'yes/no' screening assays, through semi quantitative ranking of kinetic association, dissociation and equilibrium constants for a family of binding partners, to deriving constants comparable with those which would be obtained in free solution. A dependence of the initial rate of biomolecular association on concentration allows analyte concentration to be measured--an increasingly common application class. This is often employed in situations where a rapid determination is required The ability to recover bound analyte from the sensor surface in sufficient amounts for subsequent characterization is opening up new routes to the parallel analysis of structure and function.     

Binding of alkylisocyanides with soybean leghemoglobin. Comparisons with sperm whale myoglobin

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are slower. For a given protein, the dissociation rate constants are not much different when different isocyanides are used (except for methyl), whereas the association rates show complex behavior in relation with the alkyl chain length; singular differences are observed between leghemoglobin and sperm whale myoglobin in this regard. For myoglobin, the binding rate constants decrease from methyl to n-propyl, but remain approximately the same when the ligand carries a still longer alkyl chain. In contrast, for leghemoglobin, although the rate constants decrease from methyl to n-propyl, they show a progressive and important rise with longer alkyl substituents: n-butyl and n-pentyl.     

Influence of various fluorescent and non-fluorescent labels on the kinetics of the complex formation of alpha-chymotrypsin with basic pancreatic trypsin inhibitor (Kunitz).

The association of alpha-chymotrypsin with basic pancreatic trypsin inhibitor was studied using extrinsic signals produced by fluorescent and nonfluorescent labels. The reactive dyes were covalently bound to the proteins in the complexed state, in which the binding region was protected. The signals were sufficiently large to measure the complex formation at protein concentrations of 10(-9)M by fluorescence and down to 10(-6)M by absorption. Therefore, the association and dissociation could be followed over a broad range of concentration. Good correspondence was observed between data which were obtained with different labels and with published values for the unlabeled proteins. Existing differences could be explained by different buffer conditions used by the different authors. Also the pH dependence of the dissociation rate constants was essentially unaltered by the introduction of the labels. The large signals allowed a direct measurement of the equilibrium constants of dissociation, even at high pH, at which they are in the range of 10(-8)M. The experimentally determined binding constants were in agreement with those calculated from the rate constants. The temperature dependence of the binding constants revealed a small positive and pH-dependent enthalpy change [deltaHo = 4.0 kcal/mol (16.7 kJ) at H 7.0[. The results prove that the labeling can be performed in such a way that the equilibrium and kinetic parameters of the system studied are not significantly influenced.     

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nM to 30 nM. The association and dissociation rate constants of the immobilized aptamer–thrombin complex were 6.7 × 10³ M⁻¹ s⁻¹ and 1.0 × 10⁻⁴ s⁻¹, respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.     

Comparison of high pressure-induced dissociation of single-stranded DNA-binding protein (SSB) from high pressure-sensitive and high pressure-adapted marine Shewanella species

The effects of hydrostatic pressure on protein quaternary structure were compared for recombinant single-stranded DNA-binding protein (SSB) derived from piezosensitive, piezotolerant, and obligately piezophilic ("pressure-loving") marine Shewanella strains. The pressure-induced dissociation of the oligomeric SSB proteins was investigated using fluorescence anisotropy. The SSBs all exhibited striking similarity in the pressure-dependent behavior of the fluorescence intensity and emission spectrum as well as in their dissociation constants at atmospheric pressure. The free energies of subunit association into tetramers for all SSBs were between -27 and -30 kcal mol(-1). However, SSB from the piezosensitive Shewanella strain S. hanedai was more sensitive to pressure than that of the SSB proteins from the piezotolerant or piezophilic bacteria. The volume change of association obtained from the pressure dependence of dissociation at a fixed protein concentration (Delta V(p)) for SSB from S. hanedai was 394-402 ml mol(-1). The Delta V(p) values for SSB from the deeper-living Shewanellas were smaller and ranged from 253 to 307 ml mol(-1). Differences between the primary structures of the SSB proteins that could correlate with differences in sensitivity to pressure-induced dissociation were examined.     

Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and non-specific binding.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.     

Effect of substrate on the spin state of cytochrome P-450 in hepatic microsomes

The effect of substrate on the spin state of oxidized cytochrome P-450 in liver microsomes prepared from phenobarbital-pretreated rats has been examined. Formation of the substrate-induced Type I difference spectrum was found to correlate quantitatively with the disappearance of the ferric low-spin esr signal of cytochrome P-450. The dissociation constant of substrate for oxidized cytochrome P-450 obtained by optical methods was found to be the same as that obtained from esr methods provided that the same high microsomal protein concentration was used. However, a decrease in microsomal protein concentration leads to an apparent increase in the affinity of substrate for oxidized cytochrome P-450, indicating a dependence of lipophilic substrate dissociation constants on the membrane concentration.     

Problems associated with the application of the Cheng-Prusoff relationship to estimate atropine affinity constants using functional tissue responses

We have studied problems associated with the application of the Cheng-Prusoff relationship to the estimation of atropine dissociation constants from isolated guinea-pig tracheal responses. The values obtained have been compared to dissociation constants derived using Schild analysis. It was observed that when either carbachol (an agonist of high efficacy) or pilocarpine (an agonist of low efficacy) was used the dissociation constants estimated for atropine using the Schild analysis were very similar to those estimated using the Cheng-Prusoff relationship. In these latter experiments the agonist concentration used was the EC80. When the agonist concentration used was increased to supramaximal concentrations (3-fold greater than the EC100) the dissociation constants derived were overestimations by approximately 10-fold. It is concluded that in certain circumstances the results obtained using both the Cheng-Prusoff relationship and Schild analysis are comparable. However, it is unlikely that the Cheng-Prusoff relationship is generally applicable and that Schild analysis is clearly preferable in determining antagonist dissociation constants.     

Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.     

Determination of active concentrations and association and dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification

Accurate determination of kinetic rate constants for interacting biomolecules requires knowledge of the active concentrations of the participating molecules. Also, in other biomedical and clinical applications, sensitive, precise and accurate methods are needed to determine the concentration of biologically active molecules, which frequently constitute only a fraction of the total molecular pool. Here we report a novel development of the approach to determining active concentrations based on surface plasmon resonance (SPR) technology. The method relies on changes in binding rates with varying flow rates under conditions of partial mass transport, and does not require standards of known concentrations, given that the molecular mass of the molecule of interest is known. We introduce an analytical solution to the differential equations describing the formation of a 1:1 bimolecular complex, taking into account both the association and dissociation reactions, under partial mass transport limitations. This solution can be used in global fitting to binding curves obtained at different flow rates. The accuracy, precision, and sensitivity of this approach were determined in experiments involving binding of tyrosine-phosphorylated recombinant proteins to anti-phosphotyrosine antibodies, where the active concentration could be determined independently by in vitro phosphorylation with (33)P. There was an excellent agreement between the active concentrations determined by the analytical SPR-based method and by determination of the level of radioactivity of the phosphorylated protein. The SPR-based method allows determination of protein concentrations at picomolar levels. A procedure for accurate determinations of association and dissociation rate constants, based on the analytical solution of the mass transport and binding theory, is outlined.     

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association?dissociation reaction of ApoE can be modeled with a monomer?dimer?tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association?dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer?dimer?tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.     

Steroid-protein interactions. Stopped flow fluorescence studies of the interaction between steroid hormones and progesterone-binding globulin.

Stopped flow fluorometry, measuring changes in the intrinsic fluorescence of progesterone-binding globulin (PBG), was used to determine the association and dissociation rates of the interaction of PBG with seven delta4-3-ketosteroids. The rates of formation and dissociation of the PBG-progesterone complex were measured as a function of concentration and temperature. At 20 degrees, kon = 8.7 X 10(7) M-1 S-1 and koff = 0.060 S-1. The association rate constants for progesterone, deoxycorticosterone, testosterone, testosterone acetate, and medrogestone were found to be the same within experimental error. The different affinities of PBG for these steroids result from the dissociation rate constants of the steroids which ranged from 0.43 S-1 for testosterone to 0.024 S-1 for medrogestone. Two corticosteroids, corticosterone and cortisol, were both bound somewhat more slowly (approximately 5 X 10(7) M-1 S-1). Reflecting their very low affinity for PBG both steroids dissociate very rapidly: corticosterone at 1.4 S-1 and cortisol at 90 S-1. The ratio of association to dissociation rate constants gave affinity constants in agreement with independently determined constants.     

Determination of the two-dimensional interaction rate constants of a cytokine receptor complex

Ligand-receptor interactions within the plane of the plasma membrane play a pivotal role for transmembrane signaling. The biophysical principles of protein-protein interactions on lipid bilayers, though, have hardly been experimentally addressed. We have dissected the interactions involved in ternary complex formation by ligand-induced cross-linking of the subunits of the type I interferon (IFN) receptors ifnar1 and ifnar2 in vitro. The extracellular domains ifnar1-ectodomain (EC) and ifnar2-EC were tethered in an oriented manner on solid-supported lipid bilayers. The interactions of IFNalpha2 and several mutants, which exhibit different association and dissociation rate constants toward ifnar1-EC and ifnar2-EC, were monitored by simultaneous label-free detection and surface-sensitive fluorescence spectroscopy. Surface dissociation rate constants were determined by measuring ligand exchange kinetics, and by measuring receptor exchange on the surface by fluorescence resonance energy transfer. Strikingly, approximately three-times lower dissociation rate constants were observed for both receptor subunits compared to the dissociation in solution. Based on these directly determined surface-dissociation rate constants, the surface-association rate constants were assessed by probing ligand dissociation at different relative surface concentrations of the receptor subunits. In contrast to the interaction in solution, the association rate constants depended on the orientation of the receptor components. Furthermore, the large differences in association kinetics observed in solution were not detectable on the surface. Based on these results, the key roles of orientation and lateral diffusion on the kinetics of protein interactions in plane of the membrane are discussed.     

Modeling binding kinetics at the Q(A) site in bacterial reaction centers

Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentration and solvent viscosity dependence of the association rate indicate a second-order rate-determining step. The association rate constants (k(on)) range from 10(5) to 10(7) M(-)(1) s(-)(1). Association and dissociation rate constants were determined at pH values above the hydroxyl pK(a) for five hydroxyl naphthoquinones. These negatively charged compounds are competitive inhibitors for the Q(A) site. While the neutral quinones reach equilibrium in milliseconds, anionic hydroxyl quinones with similar K(d) values take minutes to bind or dissociate. These slow rates are independent of ionic strength, solvent viscosity, and quinone concentration, indicating a first-order rate-limiting step. The anionic semiquinone, formed by forward electron transfer at the Q(A) site, also dissociates slowly. It is not possible to measure the association rate of the unstable semiquinone. However, as the protein creates kinetic barriers for binding and releasing anionic hydroxyl quinones without greatly increasing the affinity relative to neutral quinones, it is suggested that the Q(A) site may do the same for anionic semiquinone. Thus, the slow semiquinone dissociation may not indicate significant thermodynamic stabilization of the reduced species in the Q(A) site.     

Magnesium ion dependent equilibria, kinetics, and thermodynamic parameters of Artemia ribosome dissociation and subunit association

The influence of magnesium ion concentration on the equilibrium and kinetics of Artemia ribosome dissociation and subunit association has been studied by laser light scattering. Ribosomal aggregation was found to be reduced by addition of 0.1-0.05 mM spermidine and KCl concentrations of 100 mM. The ribosomes were found to be stable at low [Mg2+], and the curves obtained for ribosome-subunit equilibrium were independent of the direction and origin of the magnesium ion titration. Thermodynamic parameters were obtained from the temperature-dependent equilibria and have been compared to those of wheat germ and Escherichia coli type A ribosomes. The entropy term calculated for the association of 40S and 60S subunits is small, and the reaction is exothermic. The entropy term is negative, favoring subunit dissociation, and contributes less to the free energy than the enthalpy term. Rate constants for ribosome dissociation and subunit association have been determined. The reaction curves gave no evidence for sequential processes and were homogeneous.     

Analysis of conformationally-linked dissociation of proteins

Conformationally-linked dissociation equilibria of dimeric proteins have been examined to determine how experimentally obtainable parameters, such as the apparent dissociation constant, k_D, and the apparent conformational transition constant, K_conf, are related to intrinsic subunit interaction constants, K_A or K_B, and intrinsic isomerization constants, K₁ or k₂. Limiting models are considered in which either the conformational change occurs before dissociation or in which the dissociation occurs before the conformational change, as well as a general model including both possibilities. Models are also considered in which three conformations are allowed or in which four subunits (tetramers) are involved. Simulated data for the dimer equilibria are presented to show how variation of protein concentration and variation of certain constants affect the proportion of various molecular species, the weight-average molecular weight, and the overall extent of conformational change. Methods are suggested to distinguish between the different limiting cases based upon the dependence of K_D and/or K_conf on protein concentration, perturbant concentration, and temperature. It is concluded that methods used to calculate self-dissociation constants oligomeric proteins include linked isomerization reactions such that the equilibrium constant obtained should not be considered as a true subunit interaction term. Indeed, dissociation can occur under the influence of a perturbant with no change in the intrinsic affinity of the subunits but with the sole effect of the perturbant being on a linked conformational change. Additional experiments on the thermodynamics of the conformational change are required to determine the actual relationship. Depending on the complexity of the equilibria involved and the relative value of the equilibrium constants, the extent of the conformational change can vary directly with, vary inversely with, or he independent of the total protein concentration. Even when intrinsic subunit affinities are not affected by the perturbant, the extent of conformational change can vary with protein concentration. Interpretation of data from proteins which may be involved in conformationally-linked dissociation reactions, therefore, must be made with caution.     

Kinetic and thermodynamic characterization of the interaction between Q beta-replicase and template RNA molecules

The specific binding of the RNA polymerase Q beta-replicase to some of its RNA template molecules, the single-stranded RNA variant MDV and also Q beta-RNA, was studied under various conditions by using a gel-retardation assay as well as filter retention. The dissociation of the replicase-RNA complex proceeds with first-order kinetics. The dependence of the dissociation rate constant on the concentration of monovalent ions suggests that there are three contacts between the midivariant (MDV) RNA and the replicase. Through analysis of the temperature dependence of the dissociation rate constant, values of 35 and 43 kJ/mol were obtained for the activation energies of complex dissociation between Q beta-replicase and the minus (-) and plus (+) strands of MDV, respectively. The bimolecular association is of second order with high rate constants that increase when the temperature is raised and decrease at higher salt concentrations. The equilibrium constants vary between 4.10(11) M-1 and 5.10(7) M-1, according to the reaction conditions. The temperature dependence of Ka gives delta H = -39 kJ/mol for MDV- and -47 kJ/mol for MDV+. Under nearly all conditions, distinct differences in the association and dissociation rates of plus and minus strands of MDV are observed. The binding of the small variant MDV to Q beta-replicase is three orders of magnitude stronger than the binding of the natural template Q beta-RNA.     

Fluorescence correlation spectroscopy analysis of the dynamics of tubulin interaction with RB3, a stathmin family protein

We have used fluorescence correlation spectroscopy to analyze the interaction of GTP-tubulin with rhodamine-labeled RB3, a neural protein of the stathmin family, and to determine the kinetic pathway of the association process. RB3 displayed slow association-dissociation kinetics with tubulin depending on the square of the tubulin concentration. The values of the apparent association and dissociation rate constants of the complex of two tubulin dimers and RB3 are determined to be (3.52+/-0.14)x10(-3) micro;M(-2)/s and (1.9+/-0.6)x10(-3) s(-1) respectively. The value of the equilibrium dissociation constant for the first tubulin-RB3 interaction is estimated to be >or=7 microM at 20 degrees C.