Conformational changes in glycogen phosphorylase studied with a spin-label probe.

Phosphorylase b and a were covalently modified on essentially one -- SH group per subunit by a spin label 4-(2-iodoacetamido)2,2,6,6-tetramethyl piperidinyloxyl. The labelled enzyme is fully active and exhibits all the characteristics of the native molecule. The electron spin resonance spectrum of the label depends on the nature of the ligand that is bound to the enzyme. This property of the spin label is used to study the interaction between the enzyme (both in the b and a forms) and activators (AMP, IMP, CMP), inhibitors (ADP, ATP, UDPG, glucose 6-phosphate), substrates (phosphate and glucose 1-phosphate) and other ligands (adenosine, beta-glycerol-2-phosphate). The interactions are analysed in terms of the apparent ligand dissociation constants and the multiplicity of conformations that this regulatory enzyme exhibits.     

Minimal models of multi-site ligand-binding kinetics

Mathematical models based on the current understanding of co-operativity in ligand binding to the (macro) molecule and relating the dose-response (saturation) curve of the (macro) molecule ligation to intrinsic dissociation constants characterizing the affinities of ligand for binding sites of both unliganded and partly liganded (macro) molecule have been developed. The simplified models disregarding the structural properties and considerations concerning conformational changes of the (macro) molecule retain the ability to yield sigmoid curves of ligand binding and reflect the co-operativity. Model 1 contains only three parameters, parameter κ (a multiplier characterising the change in the affinity) reflects also the existence and type of co-operativity of ligand binding: κ<1 corresponds to positive co-operativity, κ>1 to the negative and κ=1 to the absence of any co-operativity. Model 2 contains an extra parameter, ω, equilibrium constant for the T₀↔R₀ transition but fails to produce dose-response, which would suggest negative co-operativity. For any fixed n>1, the deviation of the dose-response (saturation) curve from the Henri hyperbola depends either solely on parameter κ (Model 1) or also on parameter ω (Model 2). The (macro) molecule being a receptor, both models yield a diversity of dose-response curves due to possible variety of efficacies of the (macro) molecule. The models may be considered as extensions of the Henri model: in case the dissociation constants remain unchanged, the proposed models are reduced to the latter.     

An experimental method for the determination of enzyme-competitive inhibitor dissociation constants from displacement curves: application to human renin using fluorescence energy transfer to a synthetic dansylated inhibitor peptide

We developed a facile procedure for the determination of enzyme-competitive inhibitor dissociation constants over a wide range of potencies at any ratios of enzyme, labeled ligand, and inhibitor. The assay uses displacement curves and a fluorescent-labeled ligand to allow experimental determination of dissociation constants (Kd's) of inhibitors of human renin, a highly specific enzyme, for which numerous high affinity (up to 100 pM) inhibitors have been synthesized. The procedure involves binding a dansylated competitive inhibitor, U80215, followed by its displacement by an unlabeled inhibitor of renin. Binding of U80215 is monitored by fluorescent energy transfer from the renin tryptophans to the dansyl moiety; displacement of U80215 by an unlabeled inhibitor is monitored by a reversal of this process. The procedures may be used to determine the potencies of unlabeled inhibitors up to 100 pM affinities and to determine kinetic binding constants. The concepts described should also be useful in other protein/ligand systems.     

Quantitative affinity electrophoresis of RNA–small molecule interactions by cross-linking the ligand to acrylamide

We show that the affinity electrophoresis analysis of RNA–small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA–aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide–aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C–C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand.     

Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution

Affinity characterization by mass spectrometry (AC–MS) is a novel LC–MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC–MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC–MS was likewise extended to a larger set of soluble protein–ligand systems. It was established as a valuable resource for counter screen and structure–activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.     

Allostery in the lac operon: population selection or induced dissociation?

Allostery, the modulation of function of a protein at one site by the binding of a ligand at a different site, is a property of many proteins. Two kinetically distinct models have been proposed: i) The induced fit model in which the ligand binds to the protein and then induces the conformational change. ii) The population selection model, in which the protein spontaneously undergoes a conformational change, which is then ‘captured’ by the ligand. Using measured kinetic constants for the lac repressor the contribution of population selection vs. induced dissociation is quantified by simulating the kinetics of allostery. At very low inducer concentration, both mechanisms contribute significantly. Total induction, though, is small under these conditions. At increasing levels of induction the induced dissociation mechanism soon dominates, first due to binding of one inducer, and then from two inducers binding.     

Reactions of Mycobacterium tuberculosis truncated hemoglobin O with ligands reveal a novel ligand-inclusive hydrogen bond network

Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O(2) and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 microM(-1) s(-1)) and dissociation (<0.006 s(-1)) of O(2) are among the slowest known, precluding transport or diffusion of O(2) as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25- and 77-fold increases in the combination rate constants for O(2) and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the chi((2,1)) dihedral angle for the indole ring increases progressively from approximately 93 degrees to at least 100 degrees in going sequentially from the deoxy to CO to O(2) derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O(2) dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.     

On porcine pancreatic alpha-amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o-nitrophenylmaltoside) by inhibition studies. Correlation with the five-subsite energy profile

Hydrolysis of small substrates (maltose, maltotriose and o-nitrophenylmaltoside) catalysed by porcine pancreatic alpha-amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Non-linear double-reciprocal plots are obtained at high maltose, maltotriose and o-nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [v/(V-v)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated. Maltose and maltotriose are inhibitors of the amylase-catalysed amylose and o-nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kiapp varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s). The values of the dissociation constants Ks1 and Ki1 of the maltose-amylase complexes are identical. According to the five-subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4,5) or (3,4), maltotriose (as substrate and as inhibitor) and o-nitrophenyl-maltoside (as substrate) bind to the same three subsites (3,4,5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o-nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.     

Calorimetric determination of cooperative interactions in high affinity binding processes

It is demonstrated that isothermal titration calorimetry can be used to determine cooperative interaction energetics even for extremely tight binding processes in which the binding affinity constants are beyond the limits of experimental determination. The approach is based on the capability of calorimetry to measure the apparent binding enthalpy at any degree of ligand saturation. When calorimetric measurements are performed under conditions of total association at partial saturation, the dependence of the apparent binding enthalpy on the degree of saturation is a function only of the cooperative binding interactions. The method developed in this paper allows an independent estimation of cooperative energetic parameters without the need to simultaneously estimate or precisely know the value of the association constants. Since total ligand association at partial saturation is achieved only at macromolecular concentrations much larger than the dissociation constants, the method is especially suited for high and very high affinity processes. Biological associations in this category include fundamental cellular processes like cell surface receptor binding or protein-DNA interactions.     

Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors.

To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.     

Increased precision for analysis of protein–ligand dissociation constants determined from chemical shift titrations

NMR is ideally suited for the analysis of protein-protein and protein ligand interactions with dissociation constants ranging from ~2 μM to ~1 mM, and with kinetics in the fast exchange regime on the NMR timescale. For the determination of dissociation constants (K ( D )) of 1:1 protein-protein or protein-ligand interactions using NMR, the protein and ligand concentrations must necessarily be similar in magnitude to the K ( D ), and nonlinear least squares analysis of chemical shift changes as a function of ligand concentration is employed to determine estimates for the parameters K ( D ) and the maximum chemical shift change (Δδ(max)). During a typical NMR titration, the initial protein concentration, [P (0)], is held nearly constant. For this condition, to determine the most accurate parameters for K ( D ) and Δδ(max) from nonlinear least squares analyses requires initial protein concentrations that are ~0.5 × K ( D ), and a maximum concentration for the ligand, or titrant, of ~10 × [P (0)]. From a practical standpoint, these requirements are often difficult to achieve. Using Monte Carlo simulations, we demonstrate that co-variation of the ligand and protein concentrations during a titration leads to an increase in the precision of the fitted K ( D ) and Δδ(max) values when [P (0)] > K ( D ). Importantly, judicious choice of protein and ligand concentrations for a given NMR titration, combined with nonlinear least squares analyses using two independent variables (ligand and protein concentrations) and two parameters (K ( D ) and Δδ(max)) is a straightforward approach to increasing the accuracy of measured dissociation constants for 1:1 protein-ligand interactions.     

Kinetics and thermodynamics of ouabain binding by intact turkey erythrocytes: effects of external sodium ion, potassium ion, and temperature

The kinetics of association and dissociation for the ouabain-Na+,K+- dependent ATPase complex have been studied in intact turkey erythrocytes as a function of external Na+ concentration, K+ concentration, and temperature. At free ligand concentrations substantially exceeding the concentration of available binding sites, the association reaction exhibits pseudo-first-order kinetics with an association rate constant (k1) that is conveniently determined over a wide range of temperatures (5-37 degrees C). The dissociation reaction exhibits strict first-order kinetics with a dissociation rate constant (k-1) that has the unusual property, in the turkey cell, of being sufficiently great to permit its direct determination even at temperatures as low as 5 degrees C. Values for the equilibrium binding constant for the ouabain-ATPase complex (KA) predicted from the ratio of the association and dissociation rate constants agree closely with independently measured values of KA determined directly under conditions of equilibrium binding. KA is a sensitive function of the composition of the external ionic environment, rising with increasing Na+ concentration and falling with increasing K+ concentration. These changes in KA are shown to be quantitatively attributable to changes in the rate constant k1, k-1 in contrast being unaffected at any given temperature by even very large changes in Na+ or K+ concentration. Arrhenius plots of k1 and k-1 both yield straight lines over the entire temperature range corresponding to activation energies for association and dissociation of 29.5 and 24.2 kcal/mol, respectively. These observations have made it possible to calculate the following standard values for the ouabain binding reaction in the presence of 150 mM Na+: delta G degree = -9.8 kcal/mol; delta H degree = +5.3 kcal/mol; delta S degree = +48.7 cal/degree/mol. The large positive value of delta S degree presumably reflects a highly ordered configuration of the ouabain-free ATPase molecule that is lost upon ouabain binding and that "drives" the reaction despite the positive value of delta H degree.     

Some Thoughts about Bond Energies,Bond Lengths,and Force Constants

The bond energy (BE) of a polyatomic molecule cannot be measured and, therefore, determination of BEs can only be done within a model using a set of assumptions. The bond strength is reflected by the intrinsic BE (IBE), which is related to the intrinsic atomization energy (IAE) and which represents the energy of dissociation under the provision that the degree of hybridization is maintained for all atoms of the molecule. IBE and BE differ in the case of CC and CH bonds by the promotion, the hybridization, and the charge reorganization energy of carbon. Since the latter terms differ from molecule to molecule, IBE and BE are not necessarily parallel and the use of BEs from thermochemical models can be misleading. The stretching force constant is a dynamical quantity and, therefore, it is related to the bond dissociation energy (BDE). Calculation and interpretation of stretching force constants for local internal coordinate modes are discussed and it is demonstrated that the best relationship between BDEs and stretching force constants is obtained within the model of adiabatic internal modes. The valence stretching force constants are less suitable since they are related to an artificial bond dissociation process with geometrical relaxation effects suppressed, which leads to an intrinsic BDE (IBDE). In the case of AX_n molecules, symmetric coordinates can be used to get an appropriate stretching force constant that is related to the BE. However, in general stretching force constants determined for symmetry coordinates do not reflect the strength of a particular bond since the related dissociation processes are strongly influenced by the stability of the products formed.     

Identical independent sites for dye ligand on bovine serum albumin demonstrated by multivariate analysis

The most fundamental parameters concerning an interaction between a ligand and a protein are equilibrium constants and the number of binding sites. The Scatchard plot has for a long time been widely used to obtain those parameters. However, controversy in 1982-1983 over the reliability of this plot (the graphical estimation of the number of identical independent sites from the x-intercept) indicated that some methodologies other than the Scatchard plot are expected. Over the past decade, we have developed a method for applying multivariate analysis to the problem of determining spectral features of a ligand associated with a protein molecule. In principle, this method is based mainly on the computer-assisted adjustment of dissociation constants to an assumed reaction model. We discovered in this process that an n-parameter, introduced into an equation for calculating the amount of dye ligand bound to a protein, coincided with the number of identical independent sites, under a certain condition in principal factor analysis calculation. In this study, we established a new methodology for determining the number of identical independent sites using synthesized spectral series, and we then applied this method to a simple reaction system composed of bovine serum albumin (BSA) and bromocresol purple (BCP) anions. BSA was found to have two identical independent sites for BCP anions at pH 8.8.     

Reactions of a water soluble cobalt porphyrin with thiocyanate

Rate and equilibrium constants have been determined for the reactions of Cobalt(III)Tetra(N-methyltetrapyridyl)Porphine with thiocyanate as a function of pH. The porphyrin ligand increases the substitution rates at the Co(III) center by several orders of magnitude relative to many other Co(III) complexes. The rates of formation and dissociation of the thiocyanate complexes are also dependent on the nature of the axial ligand trans to the water molecule being removed.     

Consequences of ligand bivalency in interactions involving particulate receptors: equilibrium and kinetic studies with Sephadex-concanavalin A, butylagarose-phosphorylase b, and Fc receptor-IgG dimer interactions as model systems

Theoretical consideration is given to the interaction of a bivalent ligand with particulate receptor sites, not only from the viewpoint of quantitatively describing the binding behavior but also from that of the kinetics of ligand release upon infinite dilution of a receptor-ligand mixture. In the latter regard, a general expression is derived that describes the time dependence of the amount of ligand bound as a function of two rate constants for the stepwise dissociation of cross-linked ligand-receptor complex and a thermodynamic parameter expressing the initial ratio of singly linked to doubly linked ligand-receptor complexes. An experimental study of the interaction between Sephadex and concanavalin A is then used to illustrate application of this recommended theoretical approach for characterizing the binding behavior and dissociation kinetics of a bivalent ligand for a system in which all ligand-receptor interactions may be described by a single intrinsic association constant. Published results on the interaction of phosphorylase b with butylagarose are also shown to comply with this simplest model of the bivalent ligand hypothesis; but those for the interaction between immunoglobulin G (IgG) dimers and Fc receptors require modification of the model by incorporation of different intrinsic association constants for the successive binding of receptor sites to the bivalent ligand. These results emphasize the need to consider ligand bivalency as a potential phenomenon in studies of interactions between protein ligands and particulate receptors and illustrate procedures by which the effects of ligand bivalency may be identified and characterized.     

Potentiometric titration behavior of a copolymer of glutamic acid and alanine prepared by thermal polycondensation

The dissociation behavior of a copolymer of glutamic acid and alanine was investigated by means of potentiometric titration at various ionic strengths. From the curves of the apparent dissociation constants versus the degree of dissociation, the intrinsic dissociation constant of the carboxyl groups in the copolymer was estimated to be 3.92. This value is different from that of -linked polyglutamic acid and is similar to those of γ-linked polyglutamic acids such as bacterial poly-γ-D-glutamic acid. An analytical method of titration was used to determine the ratio of - and γ-glutamyl residues in the copolymer. From the results of this method and from the intrinsic dissociation constant, it was concluded that the glutamyl residues in this copolymer are mostly or entirely γ-linked.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces, is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes.

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitiors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces , is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

A Biacore biosensor method for detailed kinetic binding analysis of small molecule inhibitors of p38alpha mitogen-activated protein kinase

Protein kinases are emerging as one of the most intensely studied classes of enzymes as their central roles in physiologically and clinically important cellular signaling events become more clearly understood. We report here the development of a real-time, label-free method to study protein kinase inhibitor binding kinetics using surface plasmon resonance-based biomolecular interaction analysis (Biacore). Utilizing p38alpha mitogen-activated protein kinase as a model system, we studied the binding properties of two known small molecule p38alpha inhibitors (SB-203580 and SKF-86002). Direct coupling of p38alpha to the biosensor surface in the presence of a reversible structure-stabilizing ligand (SB-203580) consistently produced greater than 90% active protein on the biosensor surface. The dissociation and kinetic constants derived using this Biacore method are in excellent agreement with values determined by other methods. Additionally, we extend the method to study the thermodynamics of small molecule binding to p38alpha and derive a detailed thermodynamic reaction pathway for SB-203580. The Biacore method reported here provides an efficient way to directly and reproducibly examine dissociation constants, kinetics, and thermodynamics for small molecules binding to p38alpha and possibly other protein kinases. Immobilization in the presence of a stabilizing ligand may further represent a broadly applicable paradigm for creation of highly active biosensor surfaces.