Biomedical subjects
Publications and source records attributed to D G Myszka.
The number and diversity of surface plasmon resonance (SPR) biosensor applications continue to increase. Evolutions in instrument and sensor chip technology, experimental methodology, and data analysis are making it possible to examine a wider variety of biomolecular interactions in greater mechanistic detail. SPR biosensors are poised to make a significant impact in basic research and pharmaceutical discovery.
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BIACORE biosensors are useful for measuring reaction kinetics and calculating affinity constants for macromolecular interactions. However, one drawback with the flow system used in these instruments is that the standard injection procedures limit the amount of time available to collect association-phase data. This is especially problematic during equilibrium analysis of high affinity interactions. Using protein-DNA interactions as a model system, we demonstrate a simple method for overcoming this limitation. By placing the analyte directly into the running buffer we were able to deliver a continuous supply of protein to the sensor surfaces for greater than 12 h at a time. Complete equilibrium binding profiles were generated by changing the concentration of analyte and allowing the surface reactions to reequilibrate. Analyte concentrations were also decreased to demonstrate that the binding reactions were fully reversible. This method of analysis is a simple and convenient way of directly measuring equilibrium dissociation constants for very high affinity interactions.
The anti-cell death protein BAG-1 binds to 70-kDa heat shock proteins (Hsp70/Hsc70) and modulates their chaperone activity. Among other facilitory roles, BAG-1 may serve as a nucleotide exchange factor for Hsp70/Hsc70 family proteins and thus represents the first example of a eukaryotic homologue of the bacterial co-chaperone GrpE. In this study, the interactions between BAG-1 and Hsc70 are characterized and compared with the analogous GrpE-DnaK bacterial system. In contrast to GrpE, which binds DnaK as a dimer, BAG-1 binds to Hsc70 as a monomer with a 1:1 stoichiometry. Dynamic light scattering, sedimentation equilibrium, and circular dichroism measurements provided evidence that BAG-1 exists as an elongated, highly helical monomer in solution. Isothermal titration microcalorimetry was used to determine the complex stoichiometry and an equilibrium dissociation constant, KD, of 100 nM. Kinetic analysis using surface plasmon resonance yielded a KD consistent with the calorimetrically determined value. Molecular modeling permitted a comparison of structural features between the functionally homologous BAG-1 and GrpE proteins. These data were used to propose a mechanism for BAG-1 in the regulation of Hsp70/Hsc70 chaperone activity.
A capturing assay was used to monitor a Fab-antigen interaction using a BIACORE optical biosensor. The antigen, a truncated single-site mutant (F43V) version of the CD4 receptor, was captured onto the sensor surface using an immobilized nonneutralizing monoclonal antibody. While this assay design created an oriented antigen surface, the antigen slowly dissociated during subsequent binding of the Fab, thus complicating the binding responses. In this paper, we illustrate how binding events occurring on a decaying surface can be accurately described by globally fitting the response data to a model that accounts for the background surface decay. Support for the method was obtained by showing the equilibrium dissociation constant calculated from the kinetic rate constants (Kd = 2.20 +/- 0.01 nM) was similar to the value measured in solution using titration calorimetry (Kd = 2.6 +/- 0.5 nM). The ability to interpret rate constants from decaying surfaces significantly extends the types of experimental systems that can be quantitatively studied on optical biosensors.
Surface-based binding assays are often influenced by the transport of analyte to the sensor surface. Using simulated data sets, we test a simple two-compartment model to see if its description of transport and binding is sufficient to accurately analyze BIACORE data. First we present a computer model that can generate realistic BIACORE data. This model calculates the laminar flow of analyte within the flow cell, its diffusion both perpendicular and parallel to the sensor surface, and the reversible chemical reaction between analyte and immobilized reactant. We use this computer model to generate binding data under a variety of conditions. An analysis of these data sets with the two-compartment model demonstrates that good estimates of the intrinsic reaction rate constants are recovered even when mass transport influences the binding reaction. We also discuss the conditions under which the two-compartment model can be used to determine the diffusion coefficient of the analyte. Our results illustrate that this model can significantly extend the range of association rate constants that can be accurately determined from BIACORE.
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The HIV-1 capsid protein (CA) makes an essential interaction with the human peptidyl prolyl isomerase, cyclophilin A (CypA), that results in packaging of CypA into the virion at a CA to CypA stoichiometry of approximately 10:1. The 231 amino acid residue capsid protein is composed of an amino-terminal CypA binding domain (1 to approximately 151; CA151) and a carboxyl-terminal dimerization domain (approximately 151 to 231). We find that CypA binds dimeric CA and monomeric CA151 with identical intrinsic affinities (K[d] = 16(+/-4) microM). This result demonstrates that capsid dimerization and cyclophilin A binding are not thermodynamically coupled and suggests that the substoichiometric ratio of CypA in the HIV-1 virion results from the intrinsic stability of the CA/CypA complex. In the known co-crystal structure of the CA151/CypA complex, CypA binding is mediated exclusively by an exposed capsid loop that spans residues Pro85 to Pro93. The energetic contributions to CypA binding were quantified for each residue in this loop, and the results demonstrate that the Gly89-Pro90 dipeptide is the primary cyclophilin A recognition motif, with Pro85, Val86, His87, Ala88, and Pro93 also making energetically favorable contacts. These studies reveal that the active site of CypA, which can catalyze the isomerization of proline residues in vitro, also functions as a sequence-specific, protein-binding motif in HIV-1 replication.
Using BIAcore technology, we determined the rate constants for a protein antigen-antibody interaction that was mass transport limited on the optical biosensor. The antigen consisted of a soluble form of the human T-cell receptor CD4 (two amino terminal domains, D1D2) and the antibody was an anti-CD4 monoclonal from monkey engineered with the constant domains from human IgG1. High quality response data were obtained for this interaction by orienting the attachment of the antibody on the sensor surface and correcting for instrument artifacts with control experiments. Using numerical integration and global fitting, we demonstrate that a mass transport limited reaction was the only model of those tested that described well D1D2 binding to three different surface densities of the antibody. Statistical profiling techniques showed that the error space and correlation for the parameters in the non-linear model were essentially linear, but only when the model was simultaneously fitted to data from multiple surface densities. The "on" and "off" rate constants (1.2 x 10(-6) M-1 s-1 and 2.9 x 10(-4) s-1) determined from the kinetic analysis predict an equilibrium dissociation constant (KD = 0.24 +/- 0.01 nM) that agrees with the value measured in solution by titration calorimetry (KD = 0.2 +/- 0.1 nM). The results indicate that, although the D1D2-antibody reaction is partially controlled by mass transport on the optical biosensor, by optimizing the experimental design and analyzing data from multiple surface densities it is possible to determine accurate estimates of the intrinsic equilibrium and kinetic rate constants.
The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three mutant CD4 molecules (F43V, G47S, and A55F) and characterized their structural properties, thermostability, and ability to bind gp120. Crystallographic and thermodynamic analysis showed minimal structural alterations in the F43V and G47S mutant proteins, which have solvent-exposed mutant side chains. In contrast, some degree of disorder appears to exist in the folded state of A55F, as a result of mutating a buried side chain. Real time kinetic measurements of the interaction of the mutant proteins with gp120 showed affinity decreases of 5-fold for G47S, 50-fold for A55F, and 200-fold for F43V. Although both rate constants for the binding reaction were affected by these mutations, the loss in affinity was mainly due to a decrease in on rates, with less drastic changes occurring in the off rates. These observations suggest the involvement of conformational adaptation in the CD4-gp120 interaction. Together, the structural and kinetic data confirm that F43V is a critical residue in gp120 recognition site, which may also include main chain interactions at residue Gly-47.
We demonstrate that the interaction between myoglobin and an immobilized anti-myoglobin antibody measured on BIAcore 2000 can be described by a simple bimolecular reaction mechanism. We improved the quality of the sensor data by correcting for refractive index changes and nonspecific binding using a blank sensor surface. Applying nonlinear least squares analysis, we simultaneously fitted the association and dissociation phase data generated for a range of myoglobin concentrations injected across the antibody surface. The ability to globally fit these data to a simple binding model indicates that effects related to the sensor surface, like mass transport and the dextran matrix, did not complicate the observed binding responses. These results illustrate the potential of BIAcore to monitor macromolecular interactions in real-time and the utility of global analysis to resolve the reaction kinetics.
The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.
CD4 is a transmembrane glycoprotein expressed on T-lymphocytes. It is a receptor for class II major histocompatibility complex (MHC) molecules and for the HIV envelope glycoprotein gp120. The extracellular portion of CD4 (sCD4) is a rod-shaped molecule consisting of four domains designated D1 through D4. Denaturant-induced unfolding of sCD4 and of isolated CD4 domains, D1D2 and D3D4, was measured using both ultraviolet absorbance and fluorescence difference spectroscopy. Though both absorbance and fluorescence changes arise from changes in the solvent exposure of the intrinsic tryptophan chromophores, the unfolding curves obtained with the two techniques looked very different for sCD4 and D3D4. This dissimilarity is indicative of a greater than two-state unfolding mechanism. The global three-state fit for sCD4, which simultaneously fit both absorbance and emission data to a model with one thermodynamically stable unfolding intermediate, was significantly better than the best two-state fit, suggesting that there are two unfolding regions. Unfolding of isolated D3D4 also fit a three-state model while unfolding of isolated D1D2 fit satisfactorily to a two-state model. The unfolding of these two isolated fragments could not be summed to yield the unfolding profile of sCD4, implying that an interaction between D2 and D3 is lost by splitting sCD4 between these domains. The unfolding data of isolated D1D2 and D3D4 were used with the solvent-accessible surface area of tryptophans calculated from atomic crystal structure coordinates of human D1D2 and rat D3D4 to assign the unfolding steps. The data are consistent with a model for sCD4 unfolding wherein the one stable intermediate appears to contain only the D4 domain unfolded. The remaining three domains apparently unfold as a unit. Furthermore, interactions between domains D1, D2, and D3 appear to stabilize D4, suggesting that stabilizing interactions exist between D3 and D4 even though the unfolding of the D3D4 fragment is best fit by a three-state model. This report is the first to describe a thermodynamic basis for a wide range of biological properties implicated for CD4.
The binding kinetics recorded for many interactions using BIAcore and IAsys optical biosensors do not fit a simple bimolecular interaction model (A + B<-->AB). Three methods of analysis have been used to derive estimates for kinetic constants from such data:linearization, curve fitting using the integrated rate equation, and curve fitting using numerical integration. To test how well these methods could interpret complex binding kinetics, we generated and analyzed simulated data for two systems, one involving a two-state conformational change (A + B<-->AB<-->(AB)*) and a second involving surface heterogeneity (A + B<-->AB and A + B*<-->AB*). The linearization method assumed a simple bimolecular interaction and was inadequate at interpreting these systems as both produced complex kinetics in the association and dissociation phases. The sum of two integrated rate equations correctly modeled surface heterogeneity; but, when applied nonglobally, it fit the data from the conformational change system equally well and thus provided misleading results. Numerical integration allowed a choice of model for analysis and was therefore the only method capable of returning accurate estimates of rate constants for both complex systems. Global analysis, in combination with numerical integration, provided a stringent test of the assumed model. However, this stringency suggests that its application to experimental systems will require high-quality biosensor data.
A 56-residue polypeptide was designed to fold into a stable intramolecular antiparallel coiled coil, referred to as a coiled coil stem loop. The antiparallel orientation of the alpha-helices was dictated by the alignment of hydrophobic and ionic residues in the heptad repeat sequence (a, b, c, d, e, f, g)n. The hydrophobic core at the coiled coil interface was occupied by leucine and valine residues in heptad positions d and a' and positions a and d', respectively. The interface border positions e and g were occupied by glutamic acid in the amino-terminal helix and lysine residues in the carboxy-terminal helix. A loop segment connecting the alpha-helices began and ended with the helix-breaking residues glycine and proline. Alanine and serine residues were placed on the exposed b, c, and f positions of both helices to increase the helical propensity and solubility of the peptide, respectively. Several lines of evidence argued that the synthetic peptide made with this design folded into a stable monomeric coiled coil stem loop conformation: (1) the peptide was highly soluble in 150 mM sodium chloride and 50 mM sodium phosphate, pH 7.4; (2) the circular dichroism spectrum was alpha-helical but with relative ellipticity minima at 222 and 208 nm characteristic of a coiled coil structure: (3) the peptide exhibited an alpha-helical content near 80%, which was independent of peptide concentration and unchanged in the presence of trifluoroethanol; (4) size exclusion chromatography and sedimentation equilibrium ultracentrifuge measurements confirmed that the peptide was monomeric in aqueous solution; (5) the peptide exhibited high helical content over a wide pH range; (6) the apparent Tm for unfolding the alpha-helical structure was greater than 65 degrees C, and 3.0 M urea was required to reduce the helical structure by 50%; (7) a disulfide bond was readily formed in the monomer between the amino- and carboxy-terminal cysteine residues, confirming the antiparallel orientation of the helices; and (8) the peptide competed with fibrinogen for the GPIIbIIIa receptor indicating that the RGD residues present in the loop sequence were available for binding. This work establishes that an antiparallel alignment of alpha-helices can be achieved by designing specific hydrophobic and ionic interactions within the coiled coil. The prototype coiled coil peptide represents a sequence-simplified scaffold into which residues from alpha-helices and loops of native proteins can be inserted to form conformationally constrained mimetic recognition molecules.
Sequence-simplified coiled coil polypeptides were synthesized and their folding properties characterized in order to define the role of charged border residues at the coiled coil interface for the controlled formation of homodimer and heterodimer structures. Three peptides were designed to form parallel coiled coils with valine and leucine occupying the hydrophobic interface positions a and d, respectively, of the heptad repeat abcdefg. The polypeptide designated E/K42, with the heptad repeat sequence VSSLESK, contained glutamate and lysine in the interface border positions e and g, respectively, and was designed to form a coiled coil homodimer at neutral pH. Two other polypeptides, designated E/E35 and K/K35, have the heptad repeats VSSLESE and VSSLKSK, respectively. E/E35 contains only glutamic acid at both e and g positions; K/K35, only lysine, E/E35 and K/K35 were designed to form a stable coiled coil heterodimer when combined at neutral pH. All three polypeptides were prepared by solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography followed by size-exclusion chromatography. E/K42 formed a stable dimeric coiled coil structure as determined by circular dichroism and size-exclusion chromatography. The alpha-helical content of E/K42 was highest at neutral pH and decreased at extremes of pH. The alpha-helical structure of E/K42 at micromolar concentrations had a Tm of 62-65 degrees C and exhibited a concentration dependence of thermal denaturation consistent with dimer formation. In contrast to results with E/K42, a mixture of E/E35 and K/K35, but neither alone, forms alpha-helix at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)
Two different photoaffinity analogs of 4-hydroxy coumarin, 3-(p-azidobenzyl)-4-hydroxycoumarin (AzBHC) and 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin (AzISAHC), are being used in the identification of warfarin-binding proteins present in mammalian tissue (Myszka, D. G., and Swenson, R. P. (1990) Biochem. Biophys. Res. Commun. 172, 415-422; Myszka, D. G., and Swenson, R. P. (1991) J. Biol. Chem. 266, 4789-4797). In this study, [14C]AzBHC, but not [125I]AzISAHC, was observed to specifically label a 15,000-dalton protein present in both the microsomal and cytosolic fractions of rat liver. Pretreatment of the crude protein samples with warfarin or dicoumarol completely protected the 15-kDa protein from modification by [14C]AzBHC, indicating that this photoaffinity reagent is specifically labeling a coumarin-binding protein. 4-Hydroxycoumarin itself and AzISAHC were unable to block the incorporation of this photoaffinity probe. The 15-kDa protein was isolated by two-dimensional electrophoresis and subjected to amino-terminal sequence analysis. The first 20 amino acid residues analyzed were found to be identical with the amino-terminal sequence of rat liver fatty acid-binding protein (L-FABP) (Gordon J. I., Alpers, D. H., Ockner, R. K., and Strauss, A. W. (1983) J. Biol. Chem. 258, 3356-3363). Photoaffinity labeling and protection experiments carried out on purified preparations of L-FABP paralleled the labeling results obtained in the microsomes and cytosol, confirming that L-FABP is capable of specifically binding AzBHC, warfarin, and dicoumarol. Oleic acid, an established ligand for L-FABP, can compete with the binding of the photoaffinity probe; however, it was less effective in protecting the protein than warfarin. The specificity of labeling of crude liver fractions by warfarin photoaffinity analogs reported here as well as the high concentration of FABP in liver tissue together suggest that this protein may represent a major hepatic receptor responsible for the uptake and/or transport of various oral 4-hydroxycoumarin-based anticoagulant drugs.