RESUMEN
Several new aspects of computer-assisted molecular modeling strategies and biophysical techniques, such as fluorescence spectroscopy, circular dichroism, and absorption spectroscopy, have proved useful in the analysis and description of antibody-ligand interactions. The molecular features involved in determining the specificity of antibody-ligand interactions, such as electrostatics (e.g. partial charges, salt bridges, p-cation motifs), hydrogen-bonds, polarization, hydrophobic interactions, hydration and solvation effects, entropy, and kinetics can be identified using a battery of biophysical techniques. An understanding of these parameters is essential to our use of antibodies as tools in high throughput screening of chemical libraries for the discovery of novel compounds.
Asunto(s)
Anticuerpos/inmunología , Simulación por Computador , Modelos Moleculares , Anticuerpos/química , Ligandos , Conformación Proteica , Electricidad EstáticaRESUMEN
We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g(0) of fluorescence fluctuation experiments. The normalized variance g(0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g(0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g(0) to a binding model and allows the separation of species. We first apply g(0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g(0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding of fluorescein-labeled digoxigenin to a high-affinity anti-digoxin antibody was studied experimentally. A global analysis of the fluctuation amplitude and the fluorescence intensity not only recovered the dissociation coefficient and the number of binding sites, but also revealed the molecular heterogeneity of the hapten-antibody complex. Two species were used to model the molecular heterogeneity. We confirmed the molecular heterogeneity independently by fluorescence lifetime experiments, which gave fractional populations and molecular brightness values that were virtually identical to those of the g(0) analysis. The identification and characterization of molecular heterogeneity have far-reaching consequences for many biomolecular systems. We point out the important role fluctuation experiments may have in this area of research.
Asunto(s)
Anticuerpos Monoclonales/química , Haptenos/química , Ligandos , Proteínas/química , Sitios de Unión , Sitios de Unión de Anticuerpos , Digoxina/inmunología , Colorantes Fluorescentes , Cinética , Espectrometría de Fluorescencia/métodosRESUMEN
This article describes the use of optical spectroscopy in studying antibody-hapten interactions and in determining the equilibrium binding constants. Along with equilibrium binding data, spectroscopic tools often deliver structural information on binding-induced conformational changes of antibodies (or haptens). Structural implications of results from example antibody-hapten systems are included. Fluorescence spectroscopy has been particularly useful in the area of ligand binding, and thus steady-state fluorescence quenching and fluorescence polarization are the primary techniques under discussion. A brief description of fluorescence correlation spectroscopy is also provided. Absorption techniques, including circular dichroism, are mentioned to a lesser extent. A basic description of the mathematical models involved in the analysis of binding equilibria is provided along with references to more complete works. Simulated and experimental data are used to illustrate the various experimental protocols and the appropriate analytical methods. Typical sources of errors and experimental precautions are indicated throughout the general discussion.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Haptenos/química , Animales , Anisotropía , Dicroismo Circular , Polarización de Fluorescencia , Humanos , Espectrometría de FluorescenciaRESUMEN
Two single-chain antibodies (scFv) that bind the superpotent sweetener ligand, NC-174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, kappa) and NC10.14 (IgG, lambda). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25-amino-acid polypeptide linker, and expressing them in E. coli using the pET-11a system. The recombinant proteins were purified using Ni(2+)-NTA-agarose by virtue of a hexahistidine sequence introduced to the C-terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectra of both scFv possessed predominantly beta character, as did those of the Fab, and the near-UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent.
Asunto(s)
Acetatos/inmunología , Guanidinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Edulcorantes , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Dicroismo Circular , Clonación Molecular , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Ratones , Datos de Secuencia Molecular , Estructura Molecular , Estructura Secundaria de Proteína , Ensayo de Unión Radioligante , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Espectrometría de FluorescenciaRESUMEN
We report the predicted combining site structure of the monoclonal antibody fragment, NC10.14, which is specific for the superpotent sweetener, N-(p-cyanophenyl-N'-(diphenylmethyl) guanidine acetic acid, using computer-aided molecular modeling and experimental methods, such as fluorescence spectroscopy and circular dichroism. This is the first computer-aided modeling study on a lambda-chain antibody fragment. We have also identified the amino acids that are involved in ligand binding. Aromatic residues, L:91(W), L:96(W), and H:100G(Y) are predicted to make van der Waals contacts with the p-cyanophenyl moiety of the ligand. Residue H:56(K) is predicted to provide a counterion for the acetic acid moiety, and H:50(E) provides the negatively charged potential for interaction with the positive guanidinium group. We also make a comparison of the binding site architecture of NC10.14 with that of a related monoclonal antibody fragment NC6.8.
Asunto(s)
Acetatos/química , Anticuerpos Monoclonales/química , Guanidinas/química , Modelos Moleculares , Edulcorantes/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Sitios de Unión de Anticuerpos/genética , Simulación por Computador , Ligandos , Ratones , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
The specificity of a T cell is dictated by an alpha beta T cell receptor (TCR) that recognizes a complex of peptide and a product of the major histocompatibility complex (MHC). Recent studies have begun to characterize the affinities and kinetics of these interactions, but details of the alpha beta TCR structure and function are not known. To examine some of these issues we focus in this report on a TCR derived from the T cell clone 2C. This TCR binds to a complex of the nonapeptide QL9 and the class I MHC product Ld with the highest affinity of any known TCR/ligand interaction (KD approximately 10 (-7) M). Circular dichroism showed that a single-chain TCR (scTCR) containing linked V alpha and V beta regions from T cell 2C and refolded from Escherichia coli inclusion bodies exhibited the characteristic beta-sheet structure of immunoglobulins. A sensitive assay that is capable of detecting the interaction of soluble scTCR with peptide /MHC ligand on the surface of target cells was used to demonstrate that the peptide specificity of this scTCR reflects that of the TCR found on the surface of 2C. Analysis of several scTCR V alpha region mutants confirmed that the V alpha domain is critical for the specificity of scTCR binding. Finally, we identified some notable differences in the complementarity determining regions (CDR) of the 2C TCR compared to the CDR of previously characterized, cytochrome- specific TCR. These differences are discussed in the light of what is known about antibody binding sites, the high affinity of the 2C TCR, and the nature of the residues on QL9 that are predicted to interact with the TCR.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Cartilla de ADN/genética , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Three monoclonal antibodies (mAb) with nanomolar affinity to the superpotent trisubstituted guanidinium sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidineacetic acid were studied by circular dichroism (CD) spectroscopy. Two mAb, NC6.8 (IgG2b,kappa) and NC10.8 (IgG3, kappa) exhibited similar CD spectra, but mAb NC10.14 (IgG2b, lambda) had very different CD spectra in both far- and near-UV regions. Some of these differences may be due to effects of aromatic amino acid side chains, especially Trp and Tyr, located at the immunoglobulin intradomain surfaces. Heavy- and light-chain dissociation of reduced Fab fragments in 1 M acetic acid minimized these effects. Ligand binding changed the sign and amplitudes of the near-UV CD spectra of all three mAb. Calculation of the CD difference spectra (bound minus free) of stoichiometrically bound antibody-ligand complexes allowed us to visualize the net spectral changes. On the basis of the three-dimensional structures experimentally solved for NC6.8 and theoretical models of NC10.8 and NC10.14, we suggest that the p-cyanophenyl moiety on the sweetener ligand acts as a molecular pointer in the CD spectra and identifies contact aromatic residues in the different antibody binding pockets.
Asunto(s)
Acetatos/inmunología , Guanidinas/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Edulcorantes , Acetatos/química , Anticuerpos Monoclonales/química , Clonación Molecular , Guanidinas/química , Fragmentos Fab de Inmunoglobulinas/química , Ligandos , Datos de Secuencia Molecular , Rotación Óptica , Análisis de Secuencia de ADNRESUMEN
The problem of protein stability is addressed with spectroscopic studies of equilibrium and kinetic properties of the binding of fluorescein to high-affinity monoclonal anti-fluorescyl antibodies (Mab 4-4-20), Fab fragments, and single-chain antibodies (SCA). SCA molecules contain only the variable domains of the antibody and an amino acid linker. The influence of glycerol on the antigen binding reaction is studied by circular dichroism, fluorescence, and absorption spectroscopy. The presence of glycerol in the solvent lowers the affinity of SCA for the ligand drastically, and the affinity even decreases toward lower temperatures. This effect is not observed in Fab and Mab. Analysis of the temperature jump kinetics shows that the dissociation reaction can be modeled as a two-state transition. The CD spectra indicate that the domain structure of the SCA remains unaltered in the presence of glycerol. Therefore, it is concluded that glycerol promotes the dissociation of the two variable domains of SCA. In Fab and Mab, the constant domains provide additional stabilization of the molecular structure at the antigen binding site.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antígenos/metabolismo , Fluoresceínas/metabolismo , Reacciones Antígeno-Anticuerpo , Antígenos/química , Sitios de Unión de Anticuerpos , Dicroismo Circular , Fluoresceína , Fluoresceínas/química , Glicerol/farmacología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Soluciones , Solventes , Espectrometría de Fluorescencia , Espectrofotometría , Temperatura , TermodinámicaRESUMEN
Complementarity determining region (CDR) transplant studies were conducted between two monoclonal antibodies of distinctly different specificities (anti-fluorescein monoclonal antibody (mAb) 4-4-20 and anti-single-stranded DNA (ssDNA) mAb 04-01) which possessed nearly identical light chains but dissimilar heavy chains. The variations in binding specificities between the two immunoglobulins suggested that the active-site features of anti-fluorescein antibodies were dictated by characteristics intrinsic to the heavy chain (H-chain). To identify specific regions of the H-chain which influence the structure and function of an anti-fluorescein active site, CDR transplantation was systematically employed to convert the anti-ssDNA 04-01 antibody active site to an active site with anti-fluorescein activity. Each mAb 4-4-20 H-chain CDR (HCDR) was transplanted into the H-chain of a single-chain derivative of the 04-01 molecule. A fluorescence polarization ligand binding assay was utilized to determine the equilibrium dissociation constant, Kd, of hybrid transplant single-chain antibody HCDR1-HCDR2-HCDR3(4-4-20) for fluorescein (3.8 x 10(-7) M, indicating successful conversion of an anti-ssDNA active site to an anti-fluorescein binding site. A similar Kd (6.3 x 10(-7) M) was determined using a fluorescein fluorescence quenching assay. The transplantation results are discussed in terms of the relative contribution of each HCDR to a successful conversion in antibody specificity.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , ADN de Cadena Simple/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Secuencia de Bases , Sitios de Unión de Anticuerpos , Cartilla de ADN , Fluoresceína , Fluoresceínas , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Ratones , Ratones Endogámicos BALB C/inmunología , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
Binding interactions of various synthetic oligohomonucleotides with anti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding single-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide binding to IgG or SCA resulted in quenching of the protein's tryptophan fluorescence permitting direct assessment of ligand binding under equilibrium conditions. The effect of oligothymidylate length, (dT)n, on tryptophan quenching was evaluated. The equilibrium dissociation constants (Kd) for the binding of (dT)6 and (dT)8 were the same [(1.3 +/- 0.02) x 10(-7) M], while decreasing the length of the oligothymidylate to (dT)3 increased the Kd an order of magnitude. To assess base specificity, the comparative binding of other hexahomonucleotides was examined. Neither (dA)6 nor (dC)6 showed measurable binding, while the dissociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence lifetime quenching data correlated with the steady-state binding results and indicated that the quenching process contains both dynamic and static components. The ability of BV 04-01 to bind (dT)6 and (dG)6 nucleotides was further supported by fluorescence anisotrophy studies with fluorescein-labeled hexadeoxynucleotides. Various levels of tryptophan fluorescence quenching upon titration with oligothymidylates of different length, as well as the similar affinities for (dT)6 and (dG)6, supported the concept that the groove-type binding pocket in BV 04-01 consists of binding subsites that cooperatively adapt for efficient binding of oligonucleotides.
Asunto(s)
Autoanticuerpos/inmunología , ADN de Cadena Simple/inmunología , Oligonucleótidos/inmunología , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Autoanticuerpos/química , Polarización de Fluorescencia , Cinética , Modelos Moleculares , Poli T/inmunología , Espectrometría de Fluorescencia , Triptófano/análisisRESUMEN
Single-chain antibodies are comprised of immunoglobulin light and heavy chain variable domains joined through a polypeptide linker. A single-chain autoantibody, containing the 14-amino acid 212-polypeptide linker (GSTSGSGKSSEGKG), was constructed based on the light and heavy chain variable region gene sequences of anti-single-stranded DNA autoantibody BV04-01 (IgG2b, kappa). Following protein expression in Escherichia coli, denaturation, refolding, and affinity purification, single-chain autoantibody 04-01 binding with single-stranded DNA and poly(dT) was characterized in solid-phase and solution-phase assays. Homopolymer ligand binding results demonstrated that single-chain autoantibody 04-01 possessed anti-DNA binding properties similar to BV04-01 IgG and Fab fragments. Based on x-ray crystallographic analyses of BV04-01, site-specific mutagenesis studies were conducted on 2 residues (L32Tyr and H100aTrp) involved in aromatic stacking interactions with the middle thymidine of a (dT)3 ligand.
Asunto(s)
Anticuerpos Antinucleares/genética , ADN de Cadena Simple/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antinucleares/química , Anticuerpos Antinucleares/inmunología , Secuencia de Bases , Clonación Molecular , ADN de Cadena Simple/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conejos , Triptófano/metabolismo , Difracción de Rayos XRESUMEN
A reversible noncooperative conformational transition takes place in IgG molecule at 1 divided by 3 M urea solutions in the temperature interval 10 divided by 40 degrees C. This transition is registered as an increase of chromophores exposure and preceeds denaturation which happens at the same temperatures and with higher urea concentration.
Asunto(s)
Inmunoglobulina G/análisis , Urea/farmacología , Humanos , Conformación Proteica , EspectrofotometríaRESUMEN
Affinity of IgG to the first complement factor C1q was found out to increase in 10-30% glycol solutions. Analytical ultracentrifugation and turbidity data showed that IgG molecules do not aggregate at such concentrations of glycol. The complement-binding effect may be caused by a conformational transition in the IgG molecules.
Asunto(s)
Complemento C1/metabolismo , Pruebas de Fijación del Complemento , Glicoles de Etileno , Inmunoglobulina G/metabolismo , Animales , Cobayas , Técnicas In Vitro , Cinética , Conformación Proteica , SolucionesRESUMEN
Influence of urea on the structure of human IgG and isolated Fab and Fc-fragments was investigated by temperature-perturbation difference spectroscopy and circular dichroism. It was shown that 2M urea caused non-denaturational changes of IgG quaternary structure, localized in Fab-fragments. The same changes occur in solutions with ethylene glycol and glycerine as well. Apparently the main cause of these changes is dehydration. It is possible that the investigated effects model the conformational changes which appear in immunoglobulins after antigen binding.
Asunto(s)
Fragmentos de Inmunoglobulinas , Inmunoglobulina G , Dicroismo Circular , Desecación , Fragmentos Fab de Inmunoglobulinas , Fragmentos Fc de Inmunoglobulinas , Conformación Proteica , Desnaturalización Proteica , Espectrofotometría , Triptófano , Tirosina , UreaRESUMEN
An effect of salt concentration on the human myeloma immunoglobulin G structure was studied by means of circular dichroism, thermal perturbation difference spectroscopy and isoelectric focusing in a pH gradient created by a concentration gradient of glucose in borate buffer solution. Immunoglobulin G (K) Iva showed a significant shift of isoelectric point to the alkaline region as a result of the increase in salt concentration. The difference spectra indicated a change in the exposure of tyrosine residues as a result of increase in salt concentration. No changes in the circular dichroic spectra with salt concentration were observed between 205 and 250 nm. Spectral changes observed for the undigested immunoglobulin G molecule are more marked than those observed for the isolated Fab fragments.