Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy.

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affinity (Kd = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbance spectroscopy, and fluorescence spectroscopy, we have predicted the structure of the variable domain fragment (Fv) and identified the key residues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and complexation. Most notable are H:33W, which is responsible for ligand-induced tryptophan fluorescence quenching, H:56R, which forms a salt bridge with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligand. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 A) and the Fab-ligand complex (2.2 A). Comparisons between the theoretical model and experimental native and liganded Fab structures are made.

Absorption spectroscopy of the complexation between superpotent guanidinium sweeteners and specific monoclonal antibodies.

Spectroscopic studies of antibody-antigen interactions can provide useful information about the interactive motifs and energetics involved in the intermolecular association process. In this study we used absorption spectroscopy to examine the interactions between five different monoclonal antibodies (mAb) and four superpotent ligand sweeteners. Quantitative changes in the absorption spectra in the wavelength range of 230-800 nm were utilized for the determination of intrinsic association constants and thermodynamic parameters of the mAb-ligand complexes. The intrinsic association constants for the mAb-ligand complexes were found to be in the range of 10(7)-10(5) lM-1 and were in agreement with previous radioimmunoassay determinations. For two mAb, qualitative changes in the spectra in the 340 nm range could be identified and were related to the presence of charge-transfer interaction between the guanidinium ligand and aromatic residues in the binding site of the mAb. A charge transfer spectra was observed in mAb NC10.8 with two different sweetener ligands. The thermodynamic parameters of the ligand-mAb interactions were analyzed by van't Hoff plots and in almost all cases the reactions were found to be enthalpically driven. The determinations of intrinsic affinity and thermodynamic parameters may be useful in computer-aided molecular modelling studies of the antibody binding pocket and predicted ligand docking orientations. Antibody NC6.8 was found to react with this set of sweetener ligands in a rank order that is related to their sweetness potencies and the spectroscopic findings for NC6.8 are in agreement with the X-ray diffraction data of the Fab-ligand crystal structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence spectroscopy of monoclonal antibodies produced against the fluorescyl hapten conjugated through the xanthene ring.

Two mouse anti-fluorescyl monoclonal antibodies (mAb), clones FL43.1 and FL55.3, were produced to the fluorescein hapten, which was conjugated to the carrier protein through the 4' position of the xanthene ring. Association constants (K A) and thermodynamic parameters for both mAb were ascertained by monitoring the steady-state intrinsic and fluorescein fluorescence. Both techniques were in good agreement and gaveK A values in the 10(9) M (-1) range. Ligand-induced intrinsic fluorescence quenching showed a hypsochromic shift for mAb FL43.1, but not for FL55.3, suggesting that the ligand interacts with different tryptophan residues in each mAb. Because these mAb are directed toward the phenylcarboxylate portion of fluorescein, the different ionic and structural forms should be useful as indicators of antibody binding site pH and buffering capacity near the binding site.

Identification of residues in monoclonal antibody NC10.8 that bind to the sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidinoacetic acid by using radioligand binding, absorption and fluorescence spectroscopy, computer-aided molecular modeling, and site-directed mutagenesis.

The interactive residues for mouse mAb NC10.8, which binds a superpotent guanidinium sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidinoacetic acid with high affinity (Kd = 5 nM), were examined by using radioligand competitive binding, photoaffinity labeling, absorption and fluorescence spectroscopy, computer-aided molecular modeling, and site-directed mutagenesis. Competitive ligand analogue binding data revealed important structural features and a pH sensitivity for ligand binding. Spectroscopy of the sweetener-mAb complex revealed ligand-induced fluorescence quenching and the presence of a charge-transfer band. Site-directed mutagenesis of L:96W abolished the ligand-induced fluorescence quenching and reduced Ab affinity. The apparent Kd increased from 5 nM to more than 200 nM after such modification. A theoretical model of the Fv region was generated with use of a knowledge-based algorithm, and this model was used to identify the locations of key residues in the complementarity determining regions. These experimental and theoretical studies support the prediction that the sweetener ligand coordinates with the following residues: L:34H contacts the cyanophenyl ring, L:27DR forms a salt bridge with the acetic acid moiety, L:96W forms a pi-pi interaction with the cyanophenyl ring, and H:95E contacts the positively charged aryl nitrogen. These studies are important to our understanding of Ab-ligand specificity and may also shed light on the important chemical motifs responsible for elevated levels of sweetness potency in organic compounds.

Mechanism of allergenic cross-reactions--IV. Evidence for participation of aromatic residues in the ligand binding site of two multi-specific IgE monoclonal antibodies.

The binding sites of two IgE monoclonal antibodies (mAbs), LA2 and LB4, were examined by absorption, fluorescence spectroscopy and computer-aided molecular modeling (CAMM). Absorption spectra revealed the formation of 1:1 molecular complexes for both LA2 and LB4 with a variety of structurally different ligands. For mAb LA2, the binding constants for ligands consisting of different amino acid derivatives coupled to DNP could be divided into two groups, suggesting that certain amino acid side chains (e.g. hydrophobic) of the derivatives were a contributing feature in ligand recognition. The presence of a charge-transfer band (320-340 nm) was also observed for complexation with several different ligands, indicative of aromatic ligand interactions with mAb binding site tryptophans. CAMM studies of the Fv region for both mAb support of the empirical observations and inspection of the Fv models reveal numerous binding site aromatic residues that are likely candidates for ligand recognition and complexation. The multi-specificity of these mAbs for different ligands may be due to a multitude of interactions with aromatic residues in the binding sites.

Spectroscopic evidence for charge-transfer complexation in monoclonal antibodies that bind opiates.

Molecular complexes of four monoclonal anti-morphine antibodies (mAb) with the opiate ligands morphine, oxymorphone, and naloxone were studied using UV-VIS absorption spectroscopy. Although strong overlaps in the absorption spectra of the antibodies, ligands, and complexes were observed, a curve-fitting method was developed to correlate the absorbance with the concentration of the ligand-antibody complex. Using this technique, we determined the intrinsic association constants for the mAb with morphine to be in the nanomolar range, while association constants for oxymorphone and naloxone were in the micromolar range. These values were found to be in agreement with previous radioimmunoassay determinations. We also observed different changes in the absorbancy of the mAb upon complexation with different ligands and such changes were found to be different for all four mAb examined. Upon complexation with the ligand morphine, two of the mAb (clone numbers MOR368-21 and MOR10.5) displayed distinct charge-transfer spectral bands in the 320-nm region. These observations suggest that mAb binding site tryptophans may participate in the formation of the antibody-ligand complex and such complexation involves a charge-transfer interaction.

Spectrofluorimetric study of the intermolecular complexation of monoclonal antibodies with the high potency sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid.

Molecular complexation between a set of five monoclonal antibodies (MAbs) and a N,N',N"-trisubstituted guanidinium sweetener (TGS) was studied by monitoring the intrinsic fluorescence of the MAbs. Changes in the emission spectral properties of the MAbs were found to be related to the location of tryptophan residues in the antibody complementarity determining regions (CDRs). Two of the MAbs, NC10.10 and NC10.8, showed fluorescence quenching and hypsochromic (blue) shifts in the emission maxima upon complexation with the TGS ligand. Experiments with three other MAbs, NC10.1, NC6.8 and NC2.3, revealed only monotonic fluorescence quenching. The association constants obtained by spectroscopic techniques for the different MAb-TGS complexes were found to be comparable with those determined using a conventional RIA. The thermodynamic parameters of the MAb-TGS complexation were also examined. The intermolecular complexation was found to be exothermic for four of the five MAbs in this study. However, MAb NC2.3 was found to be an exception, in that it was associated with a small positive enthalpic change. This type of spectrofluorimetric analysis can aid in the identification of interactive residues and molecular dynamics involved in TGS recognition by this set of MAb. Such information may prove useful in understanding the molecular recognition motifs responsible for the intense taste properties of high potency guanidine sweeteners.

Gentle chemical deoxygenation of hemoglobin solutions.

Dithionite is often used to deoxygenate aqueous solutions because it reacts readily with oxygen. However, milder reducing agents, that do not ordinarily react readily with oxygen, may do so in the presence of an appropriate redox catalyst. We show that dithiothreitol reacts rapidly with oxygen in concentrated hemoglobin solutions to produce a mixture of deoxy-, met- and sulf-hemoglobin. The reaction in neutral phosphate buffer is not significantly affected by superoxide dismutase, benzoate or EDTA. However, addition of catalase or horseradish peroxidase decreases the proportions of met- and sulf-hemoglobin produced. We conclude that both hemoglobin and horse radish peroxidase accept dithiothreitol as the reducing substrate in heme catalyzed reactions with their respective oxidizing substrates (dioxygen and hydrogen peroxide). As a result, deoxy-hemoglobin suitable for physical studies can be prepared with a combination of a stoichiometric excess of dithiothreitol and a catalytic amount of horse radish peroxidase.