A rapid assay for affinity and kinetics of molecular interactions with nucleic acids

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions.     

Control of kinetics by cooperative interactions

Cooperative effects in ligand binding and dissociation kinetics are much less investigated than steady state kinetics or equilibrium binding. Nevertheless, cooperativity in ligand binding leads necessarily to characteristic properties with respect to kinetic properties of the system. In case of positive cooperativity as found in oxygen binding proteins, a typical property is an autocatalytic ligand dissociation behavior leading to a time dependent, apparent ligand dissociation rate. To follow systematically the influence of the various potentially involved parameters on this characteristic property, simulations based on the simple MWC model were performed which should be relevant for all types of models based on the concept of an allosteric unit. In cases where the initial conformational distribution is very much dominated by the R-state, the intrinsic kinetic properties of the T-state are of minor influence for the observed ligand dissociation rate. Even for fast conformational transition rates, the R-state properties together with the size of the allosteric unit and the allosteric equilibrium constant define the shape of the curve. In such a case, a simplified model of the MWC-scheme (the irreversible n-chain model) is a good approximation of the full scheme. However, if in the starting conformational distribution some liganded T-molecules are present (a few percent is enough), the average off-rates can be significantly altered. Thus, the assignment of the initial rates to R-state properties has to be done with great care. However, if the R-state strongly dominates initially it is even possible to get an estimation of the lower limit for the number of interacting subunits from kinetic data: similar to the Hill-coefficient for equilibrium conditions, a measure for "kinetic cooperativity" can be derived by comparing initial and final ligand dissociation rates.     

The kinetic equation for the chloride transport cycle of band 3. A 35Cl and 37Cl NMR study

The nature of a transmembrane transport process depends largely on the identity of the reaction that is rate-limiting in the transport cycle. The one-for-one exchange of two chloride ions across the red cell membrane by band 3 can be decomposed into two component reactions: 1) the binding and dissociation of chloride at the transport site, and 2) the translocation of bound chloride across the membrane. The present work utilizes 35 Cl NMR and 37 Cl NMR to set lower limits on the rates of chloride binding and dissociation at the saturated inward- and outward-facing band 3 transport sites (greater than or equal to 10(5) events site-1 s-1 in all cases). At both 0-3 and 37 degrees C, the NMR data specify that chloride binding and dissociation at the saturated transport sites are not rate-limiting, indicating that translocation of bound chloride across the membrane is the slowest step in the overall transport cycle. Using these results, it is now possible to describe many features of the kinetic equation for the ping-pong transport cycle of band 3. This transport cycle can be decomposed into two half-reactions associated with the transport of two chloride ions in opposite directions across the membrane, where each half-reaction is composed of sequential binding, translocation, and dissociation events. One half-reaction contains the rate-limiting translocation event that controls the turnover of the transport cycle; in this half-reaction, translocation must be slower than binding and dissociation. The other half-reaction contains the non-rate-limiting translocation event that in principle could be faster than binding or dissociation. However, when the following sufficient (but not necessary) condition is satisfied, both translocation events are slower than binding and dissociation: if the non-rate-limiting translocation rate is within a factor of 10(2) (0-3 degrees C) or 2 (37 degrees C) of the overall turnover rate, then translocation is rate-limiting in each saturated half-reaction. Thus, even though chloride appears to migrate through a channel that leads from the transport site to solution, the results support a picture in which the binding, dissociation, and channel migration events are rapid compared to the translocation of bound chloride across the membrane. In this case, chloride binding to the transport site can be described by a simple dissociation constant (KD = kappa OFF/kappa ON) rather than by a Michaelis-Menten constant (KM = (kappa OFF + kappa TRANSLOCATION)/KAPPA ON).     

Beta-1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering

The regulation of cell integrin receptors involves modulation of membrane expression, shift between different affinity states, and topographical redistribution on the cell membrane. Here we attempted to assess quantitatively the functional importance of receptor clustering. We studied beta-1 integrin-mediated attachment of THP-1 cells to fibronectin-coated surfaces under low shear flow. Cells displayed multiple binding events with a half-life of the order of 1 s. The duration of binding events after the first second after arrest was quantitatively accounted for by a model assuming the existence of a short-time intermediate binding state with 3.6 s(-1) dissociation rate and 1.3 s(-1) transition frequency toward a more stable state. Cell binding to surfaces coated with lower fibronectin densities was concluded to be mediated by single molecular interactions, whereas multiple bonds were formed <1 s after contact with higher fibronectin surface densities. Cell treatment with microfilament inhibitors or a neutral antiintegrin antibody decreased bond number without changing aforementioned kinetic parameters whereas a function enhancing antibody increased the rate of bond formation and/or the lifetime of intermediate state. Receptor aggregation was induced by treating cells with neutral antiintegrin antibody and antiimmunoglobulin antibodies. A semiquantitative confocal microscopy study suggested that this treatment increased between 40% and 100% the average number of integrin receptors located in a volume of approximately 0.045 microm(3) surrounding each integrin. This aggregation induced up to 2.7-fold increase of the average number of bonds. Flow cytometric analysis of fluorescent ligand binding showed that THP-1 cells displayed low-affinity beta-1 integrins with a dissociation constant in the micromolar range. It is concluded that the initial step of cell adhesion was mediated by multiple incomplete bonds rather than a single equilibrium-state ligand receptor association. This interpretation accounts for the functional importance of integrin clustering.     

Monitoring receptor-ligand interactions between surfaces by thermal fluctuations

We describe a new method for determining receptor-ligand association/dissociation events across the interface of two surfaces (two-dimensional binding) by monitoring abrupt decrease/resumption in thermal fluctuations of a biomembrane force probe. Our method has been validated by rigorous control experiments and kinetic experiments. We show that cellular on-rate of association can be measured by analysis of intervals from a dissociation event to the next association event (waiting times). Similarly, off-rate of molecular dissociation can be measured by analysis of intervals from an association event to the next dissociation event (bond lifetimes). Different types of molecular bonds could be distinguished by different levels of reduction in thermal fluctuations. This novel method provides a powerful tool to study cell adhesion and signaling mediated by single or multiple receptor-ligand species.     

Kinetic analysis of high affinity forms of interleukin (IL)-13 receptors: suppression of IL-13 binding by IL-2 receptor gamma chain.

Interleukin-13 (IL-13) is a pleiotropic cytokine that controls growth, differentiation, and apoptosis of immune and tumor cells. To understand the mechanisms of interaction between IL-13 and IL-13 receptors (IL-13R), and the role of the IL-2 receptor common gamma chain (gammac) in IL-13 binding and processing, we have examined IL-13 binding kinetics, dissociation/shedding, and internalization in renal cell carcinoma (RCC) cell lines. We observed a new phenomena in that the apparent rate of association, but not the dissociation, was strongly related to IL-13 concentration. We also observed cooperativity phenomena in IL-13 and IL-13R interaction in control RCC (MLneo) cells, but not in cells transfected with gammac chain (MLgammac). The number of IL-13 binding sites, the effective rate of ligand association, and the dissociation rate constants were reduced in gammac-transfected cells compared to control RCC cells. Two forms of IL-13R were detected in these cell lines, which differed in the kinetics of endocytosis and dissociation/exocytosis. Only a small fraction of bound receptors (14-24%) was rapidly internalized and the same fraction of the ligand-receptor complexes was shed and/or dissociated. The expression of gammac chain did not change any of these processes. A two independent high-affinity and moderate-affinity receptor model fit the kinetic observations in gammac-transfected cells. However, in control cells, the binding kinetics were more complicated. A mathematical model that fit a set of kinetic and steady state data in control cells was selected from a set of possible models. This best-fit model predicts that 1) two different IL-13R are expressed on the cell membrane, 2) a minor fraction of IL-13R exist as microclusters (homodimers and/or heterodimers) without exogenous IL-13, 3) high morphological complexity of the gammac-negative control cell membrane affects the cooperativity phenomena of IL-13 binding, and 4) a large number of co-receptor molecules is present, which helps keep the ligand on the cell surface for a long period of time after fast IL-13 binding and provides a negative control for ligand binding via production of the high affinity inhibitor bound to IL-13. Our data demonstrate that gammac exerts dramatic changes in the kinetic mechanisms of IL-13 binding.     

Applications of isothermal titration calorimetry – the research and technical developments from 2011 to 2015

Isothermal titration calorimetry is a widely used biophysical technique for studying the formation or dissociation of molecular complexes. Over the last 5 years, much work has been published on the interpretation of isothermal titration calorimetry (ITC) data for single binding and multiple binding sites. As over 80% of ITC papers are on macromolecules of biological origin, this interpretation is challenging. Some researchers have attempted to link the thermodynamics constants to events at the molecular level. This review highlights work carried out using binding sites characterized using x‐ray crystallography techniques that allow speculation about individual bond formation and the displacement of individual water molecules during ligand binding and link these events to the thermodynamic constants for binding. The review also considers research conducted with synthetic binding partners where specific binding events like anion‐π and π‐π interactions were studied. The revival of assays that enable both thermodynamic and kinetic information to be collected from ITC data is highlighted. Lastly, published criticism of ITC research from a physical chemistry perspective is appraised and practical advice provided for researchers unfamiliar with thermodynamics and its interpretation. Copyright © 2016 John Wiley & Sons, Ltd.     

Neurexin/Neuroligin Interaction Kinetics Characterized by Counting Single Cell-Surface Attached Quantum Dots

We report what to our knowledge is a new method to characterize kinetic rates between cell-surface-attached adhesion molecules. Cells expressing specific membrane receptors are surface-labeled with quantum dots coated with their respective ligands. The progressive diminution in the total number of surface-diffusing quantum dots tracked over time collectively reflects intrinsic ligand/receptor interaction kinetics. The probability of quantum dot detachment is modeled using a stochastic analysis of bond formation and dissociation, with a small number of ligand/receptor pairs, resulting in a set of coupled differential equations that are solved numerically. Comparison with the experimental data provides an estimation of the kinetic rates, together with the mean number of ligands per quantum dot, as three adjustable parameters. We validate this approach by studying the calcium-dependent neurexin/neuroligin interaction, which plays an important role in synapse formation. Using primary neurons expressing neuroligin-1 and quantum dots coated with purified neurexin-1β, we determine the kinetic rates between these two binding partners and compare them with data obtained using other techniques. Using specific molecular constructs, we also provide interesting information about the effects of neurexin and neuroligin dimerization on the kinetic rates. As it stands, this simple technique should be applicable to many types of biological ligand/receptor pairs.     

Interaction of phenylthiolato-(2,2'',2"-terpyridine)platinum(II) cation with DNA.

The interaction between a novel aromatic thiolato derivative from the family of DNA-intercalating platinum complexes, phenylthiolato-(2,2',2"-terpyridine)platinum(II)-[PhS(ter py)Pt+], and nucleic acids was studied by using viscosity, equilibrium-dialysis and kinetic measurements. Viscosity measurements with sonicated DNA provide direct evidence for intercalation, and show that at binding ratios below 0.2 molecules per base-pair PhS(terpy)Pt+ causes an increase in contour length of 0.2 nm per bound molecule. However, helix extension diminishes at greater extents of binding, indicating the existence of additional, non-intercalated, externally bound forms of the ligand. The ability of PhS(terpy)Pt+ to aggregate in neutral aqueous buffers at a range of ionic strengths and temperatures was assessed by using optical-absorption methods. Scatchard plots for binding to calf thymus DNA at ionic strength 0.01 (corrected for dimerization) are curvilinear, concave upward, providing further evidence for two modes of binding. The association constant decreases at higher ionic strengths, in accord with the expectations of polyelectrolyte theory, although the number of cations released per bound unipositive ligand molecule is substantially greater than 1. Stopped-flow kinetic measurements confirm the complexity of the binding reaction by revealing multiple bound forms of the ligand whose kinetic processes are both fast and closely coupled. Thermal denaturation of DNA radically alters the shapes of binding isotherms and either has little effect on, or enhances, the affinity of potential binding sites, depending on experimental conditions. Scatchard plots for binding to natural DNA species with differing nucleotide composition show that the ligand has a requirement for a single G X C base-pair at the highest-affinity intercalation sites.     

Kinetics of the interaction of N-(phosphonacetyl)-L-aspartate with the catalytic subunit of aspartate transcarbamoylase. A slow conformational change subsequent to binding

The binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) to the active sites of both the free catalytic subunit of aspartate transcarbamoylase and the intact holoenzyme causes conformational changes which have been studied extensively. However, no kinetic information has been available about the sequence of events occurring during the formation or dissociation of the complexes. Stopped flow kinetics, 31P saturation transfer NMR spectroscopy, and presteady-state kinetics were used to monitor the interaction of PALA with the catalytic subunit (or a derivative containing nitrotyrosyl chromophores which served as spectral probes). The various experimental approaches lead to a mechanism that includes a rapid binding of PALA with an "on" rate of about 10(8)M-1s-1 and an "off" rate of 28 s-1, followed by a much slower isomerization of the complex with a forward rate constant of 0.18 s-1. Analysis of the presteady-state bursts of enzyme activity when the protein is added to a mixture of substrates and PALA and of the lag in activity when the PALA complex with catalytic subunit is added to substrates yielded a rate constant for the reverse isomerization of 0.018s-1. Thus, the conformational change subsequent to PALA binding leads to a 10-fold increase in the equilibrium constant for complex formation. Stopped flow kinetic measurements of the spectral change resulting from mixing the complex of PALA and nitrated protein with native enzyme showed a slow process with a t1/2 of about 11 s, whereas 31P saturation transfer NMR experiments yielded at t1/2 of about 260 ms for the dissociation of PALA from the complex. This apparent disparity is understood in terms of the two-step binding scheme where rapid dissociation of the initial ligand X enzyme complex is measured by the NMR technique and the slow isomerization of the complex is responsible for the bulk of the stopped flow signal.     

Methods for measuring rates of protein binding to insoluble scaffolds in living cells: histone H1-chromatin interactions

Understanding of cell regulation is limited by our inability to measure molecular binding rates for proteins within the structural context of living cells, and many systems biology models are hindered because they use values obtained with molecules binding in solution. Here, we present a kinetic analysis of GFP-histone H1 binding to chromatin within nuclei of living cells that allows both the binding rate constant k(ON) and dissociation rate constant k(OFF) to be determined based on data obtained from fluorescence recovery after photobleaching (FRAP) analysis. This is accomplished by measuring the ratio of bound to free concentration of protein at steady state, and identifying the rate-determining step during FRAP recovery experimentally, combined with mathematical modeling. We report k(OFF) = 0.0131/s and k(ON) = 0.14/s for histone H1.1 binding to chromatin. This work brings clarity to the interpretation of FRAP experiments and provides a way to determine binding kinetics for nuclear proteins and other cellular molecules that interact with insoluble scaffolds within living cells.     

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.     

Comparison of glucocorticoid-binding proteins in normal and neoplastic mammary tissues of the rat.

Kinetic and molecular properties of components binding [3H]triamcinolone acetonide were studied using 105,000g supernatants of lactating mammary gland, R3230AC, and dimethylbenz[a]anthracene (DMBA) induced mammary tumors of the rat. Using a dextran-coated charcoal adsorption procedure, the relationship between specific glucocorticoid binding and protein concentration was linear in the range of 0.5-4.0 mg/reaction. These cytoplasmic macromolecules bound [3H]triamcinolone acetonide with limited capacity (50-400 fmol/mg of cytosol protein) and high affinity, Kd approximately 10(-8)-10(-9) M. Optimal binding was obtained when homogenizations were made in Tris buffers, at pH 7.4, containing monothioglycerol. Time course of association of [3H]triamcinolone acetonide and its binding sites showed maximal binding by 6-8 hr at 3 degrees which remained unchanged up to 24 hr. The rate constant of association at 3 degrees was in the range of 2-4 x 10(5) M-1 min-1. The rate constant of dissociation of bound [3H]triamcinolone acetonide could not be calculated accurately since the reaction was essentially irreversible for 5 hr at 3 degrees. Estimation of the half-life of the steroid-binding protein complexes from the Kd and the rate constant for association gave a value of 11-12 hr. From ligand specificity studies, the glucocorticoids, triamcinolone acetonide, corticosterone, cortisol, and dexamethasone competed well for [3H]triamcinolone acetonide binding sites. Progesterone, aldosterone, and the anti-glucocorticoid, cortexolone, were also good competitors while androgens and estrogens were weak inhibitors of binding. The binding compenents sedimented at 7-8 S in sucrose gradients of low ionic strength and dissociated into lower molecular weight components sedimenting at 4-5S in high ionic strength gradients. Studies in vivo using animals bearing the DMBA-induced tumor demonstrated that [3H]triamcinolone acetonide binding complexes were present in cytoplasmic and nuclear compartments. Sedimentation coefficients of the cytoplasmic and nuclear forms of these receptors labeled in vivo were 7-8S and 4-5S, respectively. These studies suggest that the molecular and kinetic binding properties of glucocorticoid receptors in neoplastic mammary tissues are similar to those of the normal mammary gland.     

Exploring buffer space for molecular interactions

The interaction of two molecules binding to each other is described by two rate parameters, the association rate parameter k(a) and the dissociation rate parameter k(d). Under standardized conditions these kinetic parameters can be determined by analysis of their interaction in an affinity-based biosensor system, such as BIACORE(R) 3000. The association rate describes the collision frequency and the attraction between two molecules and the dissociation rate describes the stability of the molecular complex. By comparing the affinity of different molecules (calculated as the quotient of the association rate parameter and the dissociation rate parameter), an estimation of specificity can be obtained. For dissociation, two different aspects can be considered-the practical aspect, where one is interested in separating the two molecules, and the informative aspect, where one is interested in the reasons for the dissociation event. This review focuses on a method that was designed to solve the practical problem of regeneration, but eventually produced a considerable amount of information about the interactions themselves.     

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.     

Biochemical and pre-steady-state kinetic characterization of the hepatitis C virus RNA polymerase (NS5BDelta21, HC-J4)

Here we report a detailed characterization of the biochemical and kinetic properties of the hepatitis C virus (HCV, genotype-1b, J4 consensus) RNA-dependent RNA polymerase NS5B, by performing comprehensive RNA binding, nucleotide incorporation, and protein/protein oligomerization studies. By applying equilibrium fluorescence titrations, we determined a surprisingly high dissociation constant (K(d)) of approximately 250 nM for single-stranded as well as for partially double-stranded RNA. A detailed analysis of the nucleic acid binding mechanism using pre-steady-state techniques revealed the association reaction to be nearly diffusion controlled. It occurs in a single step with a second-order rate constant (k(on)) of 0.273 nM(-)(1) s(-)(1). The dissociation of the nucleic acid-polymerase complex is fast with a dissociation rate constant (k(off)) of 59.3 s(-)(1). With short, partially double-stranded RNAs, no nucleotide incorporation could be observed, while de novo RNA synthesis with short RNA templates showed nucleotide incorporation and end-to-end template switching events. Single-turnover, single-nucleotide incorporation studies (representing here the initiation and not processive polymerization) using dinucleotide primers revealed a very slow incorporation rate (k(pol)) of 0.0007 s(-)(1) and a K(d) of the binary enzyme-nucleic acid complex for the incoming ATP of 27.7 microM. Using dynamic laser light scattering, it could be shown for the first time that oligomerization of HCV NS5B is a dynamic and monovalent salt concentration dependent process. While NS5B is highly oligomeric at low salt concentrations, monomers were only observed at NaCl concentrations above 300 mM. Binding of short RNA substrates led to a further increase in oligomerization, whereas GTP did not show any effect on protein/protein interactions. Furthermore, nucleotide incorporation studies indicate the oligomerization state does not correlate with enzymatic activities as previously proposed.     

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.     

Quantitative analyses of bifunctional molecules

Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.