Showing papers in "Journal of Molecular Recognition in 1999"

Improving biosensor analysis.

Abstract: The quality of optical biosensor data must be improved in order to characterize the mechanism and rate constants associated with molecular interactions. Many of the artifacts associated with binding data can be minimized or eliminated by designing the experiment properly, collecting data under optimum conditions and processing the data with reference surfaces. It is possible to globally fit high-quality biosensor data with simple bimolecular reaction models, which validates the technology as a biophysical tool for interaction analysis.

Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition.

Abstract: The principles of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) are reviewed together with the basic thermodynamic formalism on which the two techniques are based. Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system. The reason is that the parameters DeltaG, DeltaH, DeltaS, and DeltaCp obtained from ITC are global properties of the system under study. They may be composed to varying degrees of contributions from the binding reaction proper, from conformational changes of the component molecules during association, and from changes in molecule/solvent interactions and in the state of protonation.

Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies.

Abstract: The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy-chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy-chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy-chain antibodies. The presence of the heavy-chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen-binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen-binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti-idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site.

Prediction of the binding energy for small molecules, peptides and proteins.

Abstract: A fast and reliable evaluation of the binding energy from a single conformation of a molecular complex is an important practical task. Knowledge-based scoring schemes may not be sufficiently general and transferable, while molecular dynamics or Monte Carlo calculations with explicit solvent are too computationally expensive for many applications. Recently, several empirical schemes using finite difference Poisson-Boltzmann electrostatics to predict energies for particular types of complexes were proposed. Here, an improved empirical binding energy function has been derived and validated on three different types of complexes: protein-small ligand, protein-peptide and protein-protein. The function uses the boundary element algorithm to evaluate the electrostatic solvation energy. We show that a single set of parameters can predict the relative binding energies of the heterogeneous validation set of complexes with 2.5 kcal/mol accuracy. We also demonstrate that global optimization of the ligand and of the flexible side-chains of the receptor improves the accuracy of the evaluation.

Survey of the 1998 optical biosensor literature

Abstract: The utilization of optical biosensors to study molecular interactions continues to expand. In 1998, 384 articles relating to the use of commercial biosensors were published in 130 different journals. While significant strides in new applications and methodology were made, a majority of the biosensor literature is of rather poor quality. Basic information about experimental conditions is often not presented and many publications fail to display the experimental data, bringing into question the credibility of the results. This review provides suggestions on how to collect, analyze and report biosensor data.

The influence of transport on the kinetics of binding to surface receptors: application to cells and BIAcore.

Abstract: Accurate estimation of biomolecular reaction rates from binding data, when ligands in solution bind to receptors on the surfaces of cells or biosensors, requires an understanding of the contributions of both molecular transport and reaction. Efficient estimation of parameters requires relatively simple models. In this review, we give conditions under which various transport effects are negligible and identify simple binding models that incorporate the effects of transport, when transport cannot be neglected. We consider effects of diffusion of ligands to cell or biosensor surfaces, flow in a BIAcore biosensor, and distribution of receptors in a dextran layer above the sensor surface. We also give conditions under which soluble receptors can be expected to compete effectively with surface-bound receptors.

Affinity analysis of non-steady-state data obtained under mass transport limited conditions using BIAcore technology.

Abstract: Binding data obtained with Biacore instrumentation is often evaluated using a kinetic transport model where reaction rate constants and a mass transport coefficient are used to describe the interaction. Here the use of a simplified model, an affinity transport model, for determination of the affinity (KD) but not the kinetics (ka, kd) has been investigated. When binding rates were highly governed by mass transport effects the two models returned the same affinity and gave similar residuals, but ka and kd values found with the kinetic transport model were unreliable. On the other hand the affinity transport model failed to describe the data when binding curves were less influenced by mass transport effects. Under such circumstances the kinetic transport model returned correct ka and kd values. Depending on the outcome of the analysis the affinity transport model can therefore be used to reduce uncertainties of the kinetic parameters or as an easy way to determine KD values from non-steady-state data. The use of the affinity transport model is illustrated with simulated data and with binding data obtained for the interaction between a 439 Da thrombin inhibitor and immobilized thrombin. Since it is more difficult to resolve high ka values for low molecular weight analytes, the affinity transport model may be particularly useful for affinity analysis involving fast reactions between such analytes and immobilized protein targets. Copyright © 1999 John Wiley & Sons, Ltd.

A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand.

Abstract: An IgG-binding ligand library comprising 88 adsorbents based on a known lead compound (Li et al., 1998) was generated on an agarose solid phase. Individual members of the library were synthesized in two chemical steps using cyanuric chloride as the scaffold immobilized on the beaded support. The library was screened for binding of pure human IgG, whence selected ligands from the library were further assessed for specificity by the purification of IgG from human plasma. The potential of this strategy for the rapid identification and evaluation of chemical leads was demonstrated by the discovery of ligands with IgG binding capabilities. It was found that ligands comprising 3-aminophenol and an aminonaphthol moiety substituted on a triazine nucleus generally performed better than other ligands in the library. An immobilized ligand 22/8 adsorbent was able to purify IgG with high yield and a purity >99% from diluted human plasma.

Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster.

Abstract: A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site-specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha-helical structure for the main contact area.

On the protein-protein diffusional encounter complex.

Abstract: When two proteins diffuse together to form a bound complex, an intermediate is formed at the end-point of diffusional association which is called the encounter complex. Its characteristics are important in determining association rates, yet its structure cannot be directly observed experimentally. Here, we address the problem of how to construct the ensemble of three-dimensional structures which constitute the protein–protein diffusional encounter complex using available experimental data describing the dependence of protein association rates on mutation and on solvent ionic strength and viscosity. The magnitude of the association rates is fitted well using a variety of definitions of encounter complexes in which the two proteins are located at up to about 17 A root-mean-squared distance from their relative arrangement in the bound complex. Analysis of the ionic strength dependence of bimolecular association rates shows that this is determined to a greater extent by the (protein charge) – (salt ion) separation distance than by the protein–protein charge separation distance. Consequently, ionic strength dependence of association rates provides little information about the geometry of the encounter complex. On the other hand, experimental data on electrostatic rate enhancement, mutation and viscosity dependence suggest a model of the encounter complex in which the two proteins form a subset of the contacts present in the bound complex and are significantly desolvated. Copyright © 1999 John Wiley & Sons, Ltd.

Characterization of protein-glycolipid recognition at the membrane bilayer

Abstract: A growing number of important molecular recognition events are being shown to involve the interactions between proteins and glycolipids. Glycolipids are molecules in which one or more monosaccharides are glycosidically linked to a lipid moiety. The lipid moiety is generally buried in the cell membrane or other bilayer, leaving the oligosaccharide moiety exposed but in close proximity to the bilayer surface. This presents a unique environment for protein-carbohydrate interactions, and studies to determine the influence of the bilayer on these phenomena are in their infancy. One important property of the bilayer is the ability to orient and cluster glycolipid species, as strong interactions in biological systems are often achieved through multivalency arising from the simultaneous association of two or more proteins and receptors. This is especially true of protein-carbohydrate binding because of the unusually low affinities that characterize the monovalent interactions. More recent studies have also shown that the composition of the lipid bilayer is a critical parameter in protein-glycolipid recognition. The fluidity of the bilayer allows for correct geometric positioning of the oligosaccharide head group relative to the binding sites on the protein. In addition, there are activity-based and structural data demonstrating the impact of the bilayer microenvironment on the modulation of oligosaccharide presentation. The use of model membranes in biosensor-based methods has supplied decisive evidence of the importance of the membrane in receptor presentation. These data can be correlated with three-dimensional structural information from X-ray crystallography, NMR, and molecular mechanics to provide insight into specific protein-carbohydrate inter--actions at the bilayer.

Advances in surface plasmon resonance biomolecular interaction analysis mass spectrometry (BIA/MS)

Abstract: Ongoing, worldwide efforts in genomic and protein sequencing, and the ability to readily access corresponding sequence databases, have emphatically driven the development of high-performance bioanalytical instrumentation capable of characterizing proteins and protein–ligand interactions with great accuracy, speed and sensitivity. Two such analytical techniques have arisen over the past decade to play key roles in the characterization of proteins: surface plasmon resonance biomolecular interaction analysis (SPR-BIA) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). SPR-BIA is used in the real-time investigation of biomolecular recognition events, and is thereby capable of providing details on the association and dissociation kinetics involved in the interaction, information ultimately leading to the determination of dissociation constants involved in the event. MALDI-TOF is used in the structural characterization, identification and sensitive detection of biomolecules. Although the two techniques have found many independent uses in bioanalytical chemistry, the combination of the two, to form biomolecular interaction analysis mass spectrometry (BIA/MS), enables a technique of analytical capabilities greater than those of the component parts. Reviewed here are issues of concern critical to maintaining high-levels of performance throughout the multiplexed analysis, as well as examples illustrating the potential analytical capabilities of BIA/MS. Copyright © 1999 John Wiley & Sons, Ltd.

Conformational homogeneity in molecular recognition by proteolytic enzymes.

Abstract: Crystal structures for several hundred protease-inhibitor complexes have been analysed and their superimpositions have been used to demonstrate a universal relationship between inhibitor/substrate conformation and molecular recognition by all aspartic, serine, cysteine and metallo proteases Proteases universally recognize an extended beta strand conformation in all their peptidic (and non-peptidic) inhibitors and substrate analogues without significant exceptions This conformational homogeneity is illustrated here for a subset of 180 protease-inhibitor structures which are displayed as (a) structural overlays of multiple inhibitors for each of eight aspartic, eight serine, six metallo and five cysteine proteases; (b) single inhibitors each bound to different proteases; and (c) Ramachandran plots of peptide or pseudo-peptide dihedral angle pairs which demonstrate beta strands (Phi -54 degrees to -173 degrees, Psi 24 degrees to 174 degrees ) like those normally found paired in proteins as beta sheets However, unlike beta sheets, alpha and 3(10) helices, beta and gamma turns, where the folded main chain amide components are intramolecularly hydrogen bonded and thus unavailable for interaction with proteins, an inhibitor/substrate in an isolated beta strand conformation provides maximum exposure of its hydrogen bonding donors/acceptors and side chain components to a putative protease receptor This analysis highlights the advantages of a strand conformation over other elements of secondary structure for protease recognition and may lead to generic strategies for inhibitor design

The use of biosensor technology for the engineering of antibodies and enzymes

Abstract: Recently developed scientific instrumentation featuring surface plasmon resonance detection allows the detection of biomolecular interactions in real time and without chemical modification of the binding partners. These biosensors are proving invaluable tools in protein engineering, particularly in research aimed at the isolation and improvement of protein binders and catalysts from macromolecular repertoires containing billions of individual members. This article reviews the use of biosensor technology for the isolation and characterization of engineered antibodies and enzymes.

Exploring buffer space for molecular interactions.

Abstract: 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.

Receptins: a novel term for an expanding spectrum of natural and engineered microbial proteins with binding properties for mammalian proteins.

Abstract: A new term 'receptin', derived from recipere (lat.), is proposed to denote microbial binding proteins that interact with mammalian target proteins. An example of such a 'receptin' is staphyloccocal protein A which binds to the Fc part of many mammalian immunoglobulins. Several other types of 'receptins' are listed. This term may easily be distinguished from the similar term 'receptor', describing a binding site on a cell surface, mostly eukaryotic, where a secondary effect is induced inside the cell upon binding to a ligand. A receptin, however, does not necessarily have to induce a secondary event. Receptins include so called MSCRAMMs, adhesins, and also engineered receptins, affibodies, and engineered ligands. It denotes any protein of microbial origin, cell-bound or soluble, which can bind to a mammalian protein. It fulfills the need for an umbrella terminology for a large group of binding structures. In contrast, the term 'lectin' represents a group of proteins with affinity for carbohydrate structures. The new term 'receptin' includes a number of key microbial proteins involved in host-parasite interactions and in virulence. Some receptins are promising vaccine candidates.

Biosensor analysis of the interleukin-2 receptor complex.

Abstract: Surface plasmon resonance (SPR) biosensor technology has been a significant addition to the evolution and refinement of methods to study macromolecular interactions. Prior to the advent of SPR, we employed a variety of biochemical and biological techniques to study the interleukin-2/interleukin-2 receptor system (IL-2/IL-2R). By combining site-directed mutagenesis, equilibrium and kinetic radioligand binding, and competitive biological assays, we and others had begun to understand many aspects of the structure-activity relationships of the IL-2/IL-2R system. Due to the complexity of the IL-2R, cell-based assays proved limited in their ability to provide quantitative information on the binding characteristics of subclasses of the IL-2 receptor. SPR technology promised to be a new and powerful approach to the quantitative analysis of complex receptor systems. To demonstrate the feasibility of this technology, we employed Biacore analysis to investigate the ligand binding characteristics of novel, pre-assembled, IL-2R coiled-coil complexes. The results of these studies, although limited by instrumentation and data analysis, clearly established the utility of this method. Subsequently, by incorporating advancements in both of these areas, we have been able to carry out detailed kinetic analyses of the binding properties of individual IL-2R subunits as well as heteromeric complexes on the surface of a biosensor. Therefore, SPR biosensor analysis combined with other established analytical methods has proven to be a powerful tool for the analysis of complex hematopoietic receptor systems. Published in 1999 by John Wiley & Sons, Ltd.

Design, synthesis and characterisation of affinity ligands for glycoproteins.

Abstract: The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein–carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar-binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine-based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself. Copyright © 1999 John Wiley & Sons, Ltd.

The ion channel switch biosensor

Abstract: A biosensor technology is described which provides a direct measurement for functional molecular interactions, at the surface of a tethered bilayer membrane, through the electrical transduction of chemically modified ion-channels. High sensitivity of analyte detection is achieved due to the large flux of ions transmitted through the ion channel. The biomimetic sensor surface allows the molecular recognition to be measured in complex biological matrices (such as blood and sera) without compromising sensitivity. We have used the sensor for activity and concentration measurements for a range of analytes, which include bacteria, DNA, proteins and drugs. We have a quantitative model for the biosensor performance which is described by three-dimensional molecular interactions with the membrane surface and two-dimensional molecular interactions within the tethered bilayer. Copyright © 1999 John Wiley & Sons, Ltd.

Targetting of the N-terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric ScFvs blocks the E6-mediated degradation of cellular p53.

Abstract: The E6 protein of cancer-associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6-mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N-terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6-mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N-terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein.

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions.

Abstract: The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.

Active concentration measurements of recombinant biomolecules using biosensor technology.

Abstract: Whereas the concentration of a biomolecule simply refers to the amount of chemical substance per unit of volume, its active concentration refers to a relational parameter that has meaning only with respect to the molecule's ability to interact specifically with one particular ligand. When proteins are studied in a biological context, it is the biologically active concentration that is relevant, and not the total concentration of correctly and incorrectly folded molecules. Using a biosensor instrument the concentration of active biomolecules in a preparation can be measured by injecting the preparation at different flow rates onto a sensor chip surface presenting a high concentration of a specific ligand. The method can be used under conditions of partial mass transport limitation and does not require a pre-established standard curve. When the method was used to measure the active concentration of several recombinant proteins it was found that the active concentration was much lower than the nominal concentration determined by conventional methods. The active concentration also depended on the ligand used in the binding assay, reflecting the fact that active concentration can only be defined with respect to one specific probe. Such discrepancies in concentration values, if undetected, may lead to erroneous conclusions regarding the properties and behaviour of recombinant proteins tested in different assays. Copyright © 1999 John Wiley & Sons, Ltd.

Evidence for conformationally different states of interleukin‐10: binding of a neutralizing antibody enhances accessibility of a hidden epitope

Abstract: We present the mapping of two anti-human interleukin-10 (hIL-10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL-10-derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L-amino acids. The epitope-derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody-hIL-10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X-ray structure of IL-10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL-10 can adopt a conformational state other than that observed in the crystal structure.

Comparison of the three-dimensional structures of a humanized and a chimeric Fab of an anti-gamma-interferon antibody.

Abstract: The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use.

Molecular recognition events involved in the activation of the growth hormone receptor by growth hormone

Abstract: Binding of growth hormone (GH) to its receptor (GHR) is a well-studied example of molecular recognition between a cytokine and its receptor. Extensive mutagenesis studies and several crystal structures have defined the key interactive amino acid residues that are involved in binding and subsequent receptor dimerization. This review encompasses each of the three molecular recognition events involved in GHR activation, namely binding of GH to its two receptors and the interactions that occur between these receptors, Particular attention is given to species and ligand specificity of hormone binding and to the molecular recognition events involved in receptor activation, including the possibility that a conformational change in the receptor is required. Copyright (C) 1999 John Wiley & Sons, Ltd.

Unraveling the effect of changes in conformation and compactness at the antibody V(L)-V(H) interface upon antigen binding.

Abstract: We have analyzed conformational changes that occur at the interface between the light (V(L)) and heavy (V(H)) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three-dimensional structures to evaluate changes in compactness of the V(L)-V(H) interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium-sized nonprotein antigens cause an opening of the V(L)-V(H) interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the V(L)-V(H) interface. The largest changes in the atomic buried contact surface area at the V(L)-V(H) interface occur in residue pairs providing two 'shock absorbers' between the edge beta-strands of the V(L) and V(H) beta-sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the V(L)-V(H) interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the V(L)-V(H) interface can be used to change antibody binding affinity with antigens. Due to the tightly packed V(L)-V(H) interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity.

Processing of PDGF gene products determines interactions with glycosaminoglycans.

Abstract: The platelet derived growth factor (PDGF), a mitogen for mesenchymal cells, may be bound to and inhibited by heparin and other glycosaminoglycans. PDGF is a homo- or heterodimer of A- and B-chains. They occur as short (A109 and B110) and long (A125 and B160) isoforms. The latter contain basic carboxyl-terminal extensions. Dimeric A125 binds to heparin through its basic extension in a two-step reaction. The mechanism involves a conformational change and is consistent with a Monod–Wyman–Changeux allosteric model. Previous indirect experiments suggested that three critical amino acids (basic R111, K116 and polar T125) might be involved. Here, direct binding experiments using dimeric full-length mutants in surface plasmon resonanse analysis showed that all three critical amino acids in an R(X)4K(X)8T-motif contributed in a concerted manner to the high affinity binding. Mutations of these amino acids to alanine resulted in large thermodynamic changes, loss of the allosteric mechanism and order(s) of magnitude lower binding affinity. The binding mechanism and affinity of long dimeric rB were similar to the mutants. Short dimeric rA109 and rB110 showed 100 times lower binding affinity than rA125. Consequently, interactions with glycosaminoglycans in tissues varies between PDGF isoforms and may influence their local accumulation and activity. Copyright © 1999 John Wiley & Sons, Ltd.

Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester

Abstract: Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580-593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl(2) to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp-Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG(1) antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp-Phe and Phe-Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG(4) antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints.

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13‐DE1

Abstract: Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti-FITC antibody, B13-DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13-DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti-fluorescein antibody or with polyclonal anti-fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross-reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules. Copyright © 1999 John Wiley & Sons, Ltd.