Pseudo-native motifs in the noncovalent heme-apocytochrome c complex. Evidence from antibody binding studies by enzyme-linked immunosorbent assay and microcalorimetry.

When beef heart apocytochrome c is unfolded, it folds upon noncovalent heme binding (Dumont, M. E., Corin, A. F., and Campbell, G.A. (1994) Biochemistry, 33, 7368-7378). Here, the conformation of the heme-apocytochrome noncovalent complex is compared with that of holocytochrome c. A purification method was designed for obtaining in large amounts apocytochrome c that was shown by amino acid analysis and mass spectroscopy to be chemically intact. The apoprotein and its noncovalent complex were characterized by absorption, fluorescence, circular dichroism, and sedimentation velocity, confirming previous reports. Sedimentation-diffusion equilibrium showed that the apoprotein and its noncovalent complex with heme were monomeric. Surprisingly, whereas apocytochrome c was quite soluble, the noncovalent complex slowly formed heavy aggregates, thus precluding experiments at the concentrations needed for structural studies. Two monoclonal antibodies that bind strongly to distinct antigenic sites on native holocytochrome were used to probe the noncovalent complex conformation. For both antibodies, the affinity for the noncovalent complex was only about 5-10-fold smaller than that for native holocytochrome c, and about 50-100-fold larger than that for apocytochrome c. These results indicate that the noncovalent complex, although not entirely native, carries some pseudo-native structural motifs.

Conformational changes in the 20S proteasome upon macromolecular ligand binding analyzed with monoclonal antibodies.

Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).

Does a peptide bound to a monoclonal antibody always adopt a unique conformation?

The conformation of a synthetic undecapeptide derived from the Escherichia coli tryptophan synthase beta2 subunit was studied by NMR spectroscopy when bound to a monoclonal antibody (mAb 164-2) Fab' fragment directed against the native protein. The peptide 1(H-G-R-V-G-I-Y-F-G-M-K)11, peptide 11, was recognized by the antibody and its corresponding Fab' fragments with high affinity (K(D) = 1.1+/-0.2* 10(-8) M). Peptide 11 was labelled with 15N and its structure at the binding site of the Fab' 164-2 fragment was studied by isotope-editing techniques. 1H-15N heteronuclear spectra indicated the presence of two Fab'-peptide 11 complexes with two different conformations in slow chemical exchange on the chemical shift time scale.

Recognition of E. coli tryptophan synthase by single-chain Fv fragments: comparison of PCR-cloning variants with the parental antibodies.

The use of a recombinant antibody fragment instead of a complete antibody, as a conformational probe for protein structure and folding studies, can be technically advantageous provided that the recombinant fragment and its parental antibody recognize the antigen through the same mechanism. Monoclonal antibodies mAb19 and mAb93 are directed against the TrpB2 subunit of Escherichia coli tryptophan synthase and they have been extensively used as conformational probes of this protein. DNA sequences coding for single-chain variable fragments (scFv) of mAb19 and mAb93 were cloned and assembled by reverse transcription of the mRNAs from hybridomas and PCR amplification. A specialized plasmid vector, pFBX, was constructed; it enabled to express the scFvs as hybrids with the maltose-binding protein (MalE) in E. coli, and to purify them by affinity chromatography on cross-linked amylose. Six independent clones were sequenced for each hybridoma. All of them had differences in their nucleotide and amino acid sequences. A competition ELISA and the BIAcore biosensor apparatus were used to compare the energetics and kinetics with which the parental antibodies and the hybrids bound TrpB2. The antigen binding properties of the hybrids were close to those of the parental antibodies and they were only weakly affected by the differences of sequence between the clones, with one exception. The stability of one of the hybrids and its antigen binding properties were strongly modified by a change of Gln6 into Glu, introduced into its VH domain by the PCR primers. Simple models of bimolecular interaction did not fully account for the kinetic profiles obtained with the parental antibodies and the hybrids, and this complexity suggested the existence of a conformational heterogeneity in these molecules.

Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay.

A reliable ELISA based method has been developed for measuring in solution antigen/antibody association rate constants. Its rationale is as follows: antigen and antibody are mixed in solution to initiate the association. At different time intervals aliquots are withdrawn to determine by an indirect ELISA the amount of free antibody that remains in solution. The disappearance of the free mAb reflects the time course of the association reaction. To test the validity of this method, the association rate constant of a monoclonal antibody for its antigen was measured and compared with that obtained previously by a method using fluorescence. The good agreement between the results obtained with the ELISA-based method and those obtained previously by fluorescence measurement indicates that the method described permits determination of true association rate constants in solution. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or the antigen, and it can be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.

Nascent chains: folding and chaperone interaction during elongation on ribosomes.

Monoclonal antibodies that detect folding intermediates in vitro were used to monitor the appearance of folded polypeptide chains during their synthesis on the ribosomes. Nascent immunoreactive chains of the bacteriophage P22 tail-spike protein and of the Escherichia coli beta 2 subunit of tryptophan-synthase were thus identified, suggesting that they can fold on the ribosomes. Moreover, the immunoreactivity of ribosome-bound tryptophan-synthase beta-chains of intermediate lengths was shown to appear with no detectable delay compared to their synthesis. This suggested that beta-chains start folding during their elongation on the ribosomes. However, newly synthesized incomplete beta-chains were shown to interact with chaperones while still bound to the ribosome. Because of the peculiar properties of the epitope recognized by the anti-tryptophan-synthase monoclonal antibody used, it could not be concluded whether the immunoreactivity of the nascent beta-chains resulted from their ability to fold cotranslationally or from their association with chaperones which might maintain them in an unfolded, immunoreactive state.

Importance of residues 2-9 in the immunoreactivity, subunit interactions, and activity of the beta 2 subunit of Escherichia coli tryptophan synthase.

The epitope recognized by a monoclonal antibody (mAb19) directed against the beta 2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the beta chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 x 10(9) M-1, the affinity of mAb19 for native beta 2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of beta 2 (Murry-Brelier, A., and Goldberg, M.E. (1988) Biochemistry 27, 7633-7640). A mutant producing beta chains lacking residues 1-9 (beta delta 1-9) was prepared. The beta delta 1-9 protein was able to fold into a heat stable homodimer resembling wild type beta 2. Isolated beta delta 1-9 had no detectable enzymatic activity. It could bind alpha chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the beta delta 1-9 protein could bind alpha chains much more strongly and generate a 50% active enzyme. Thus, although having little role in the overall folding and stability of the protein, the 1-9 sequence of the beta chain appears strongly involved in the alpha-beta interactions and in the enzymatic activity.

In vitro and ribosome-bound folding intermediates of P22 tailspike protein detected with monoclonal antibodies.

It remains unclear whether polypeptide chains renaturing in vitro from strong denaturants proceed through the same folding pathway as chains released from ribosome within cells. Folding intermediates formed both in vivo and in vitro have been examined using three monoclonal antibodies shown previously to recognize different epitopes of the native P22 tailspike protein (Friguet, B., Djavadi-Ohaniance, L., Haase-Pettingell, C. A., King J., and Goldberg, M. E. (1990) J. Biol. Chem. 265, 10347-10351). The tailspike protein was reconstituted from polypeptide chains unfolded by urea as described by Fuchs et al. (Fuchs, A., Seiderer, C., and Seckler, R. (1991) Biochemistry 30, 6598-6604), and the appearance of immunoreactive forms during the refolding was monitored. The three antibodies discriminated intermediates at different stages in the folding pathway. On the basis of the reconstitution pathway determined from spectroscopic and hydrodynamic measurements by Fuchs et al. (1991), monoclonal antibody (mAb) 236-3 recognized partially folded monomers, mAb 155-3 recognized folded protomers in a protrimer species, and mAb 33-2 recognized the native trimer. The kinetics of appearance of the immunoreactive forms during the in vitro refolding of the protein in crude extracts of phage-infected cells was similar to that observed with the pure tailspike. Thus, the antibodies provided probes for the chain folding and association pathway in vivo. The conformation of the ribosome-bound tailspike polypeptide chains of the infected cells was analyzed with the three antibodies. The antibodies recognizing native trimer and the protrimer did not bind chains associated with the ribosomes. Antibody 236-3, which recognized structured monomers in vitro, bound to the polypeptide chains still associated with ribosomes. This result suggests that steps that take place in solution during in vitro refolding may occur in a ribosome-bound state in vivo.

Measurement of the dissociation rate constant of antigen/antibody complexes in solution by enzyme-linked immunosorbent assay.

A reliable, convenient ELISA based method has been developed for measuring the dissociation rate constants of antigen/antibody complexes in solution. Its rationale is as follows: a solution containing the preformed antigen/antibody complex is diluted well below the equilibrium dissociation constant to initiate the dissociation and, at various times after the dilution, the amount of dissociated antibody contained in an aliquot is determined by a classical ELISA, using a brief incubation of the solution in antigen coated wells. To test the validity of this method, the dissociation rate constants for several antigen/antibody complexes were compared with those obtained by classical fluorescence based methods. The good agreement between both sets of data validates the ELISA procedure. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or antigen, and it can, in principle, be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.

Nicking of the tryptophan synthase beta 2-subunit at Glu-296 prevents the conformational change undergone on binding the alpha-subunit.

Using a monoclonal antibody as conformational probe it has been shown that the weakly active nicked-beta 2 dimer of tryptophan synthase generated by proteolytic cleavage at Glu-296, does not undergo on association with alpha subunit a conformational change known to occur in intact beta 2 subunit. This alpha induced conformational change is also prevented in intact beta 2 by the coenzyme pyridoxal-5'-phosphate when the substrate L-serine is absent.

Methods for measurement of antibody/antigen affinity based on ELISA and RIA.

Various enzyme-linked immunosorbent assay or radioimmunoassay methods are currently used to quantify the antibody-antigen interaction. Only those based on indirect competition--enzyme-linked immunosorbent assay or radioimmunoassay--can provide the real thermodynamic affinity of the antibody for its antigen. They can be applied to a variety of experimental situations, some of which are reviewed here.

In vitro gene expression for the localization of antigenic determinants: application to the E. coli tryptophan synthase beta 2 subunit.

Gene expression of the beta subunit of E. coli tryptophan synthase in an E. coli cell-free transcription-translation system proceeds by pauses and produces a discrete but quite continuous pattern of nascent chains starting from the N terminus and ranging in size up to the 44 kDa end product corresponding to the completed beta chains. Using specific immunoadsorption of [35S]Met radiolabelled nascent chains by different monoclonal antibodies directed against the beta 2 subunit of E. coli tryptophan synthase, the size of the smallest N-terminal fragment reacting with each antibody has been determined by SDS electrophoretic analysis of the immunoadsorbed polypeptides. The immunoadsorption assay is performed in solution under conditions avoiding the usual drawbacks of solid phase immunoassay. This approach, in combination with the results obtained with a DNA fragment library permitted us to localize the antigenic determinants recognized by the monoclonal antibodies. The proposed method could help to localize rapidly the C-terminal boundary of an epitope, before starting systematic and precise mapping by other approaches. Moreover, the method described may be of general interest for the rapid production of a large set of C-terminal truncated polypeptides for studies of antigen-antibody recognition.

Folding on the ribosome of Escherichia coli tryptophan synthase beta subunit nascent chains probed with a conformation-dependent monoclonal antibody.

Experimental analysis of protein folding during protein synthesis on the ribosome is rendered very difficult by the low concentration of nascent polypeptides and the heterogeneity of the translation mixture. In this study, an original approach is developed for analysing nascent polypeptide structures still carried by the ribosome. Folding on the ribosome of nascent chains of the beta subunit of Escherichia coli tryptophan synthase was investigated using a monoclonal antibody (mAb 19) recognizing a conformation-dependent antigenic determinant. Upon synthesis of beta subunits in an E. coli coupled transcription-translation system, it is shown that ribosome-bound nascent polypeptides can react with the monoclonal antibody provided their size is above 11.5 kDa, which is smaller than that of both the N-terminal proteolytic and crystallographic domains (29 and 21 kDa, respectively). The gene fragments coding only for the 11.5 kDa polypeptide, with and without stop codon at the end of the corresponding mRNAs, were constructed and expressed in a cell-free wheat germ translation system. It is shown that antibody 19 reacts with this polypeptide either bound to the ribosome or free in solution. That the 11.5 kDa polypeptide acquires a condensed structure is shown by gel filtration in native conditions and by urea gradient gel electrophoresis. Moreover, it is demonstrated that this condensed structure resembles that of native beta 2 in the vicinity of the epitope for antibody 19. Indeed, the affinity of antibody 19 for the 11.5 kDa fragment, either free or bound to the ribosome, was measured (6 x 10(8) M-1) and shown to be close to that for native beta 2. It is therefore proposed that the polypeptide chain may start to fold during its biosynthesis and that, even before the appearance of an entire domain, a folded intermediate is formed that already exhibits some local structural features of the native state and of an immunoreactive intermediate previously detected during the in vitro refolding of denatured complete beta chains.

Peptide/antibody recognition: synthetic peptides derived from the E. coli tryptophan synthase beta 2 subunit interact with high affinity with an anti-beta 2 monoclonal antibody.

Recent studies have shown that the antigenic determinant recognized by a monoclonal antibody (mAb 164-2) elicited against the beta 2 subunit of E. coli tryptophan synthase is localized between residues 276 and 297 of this protein. In order to delineate more precisely the epitope recognized by this antibody, peptides ranging in length from 11 to 29 amino acids and belonging to this region were synthesized, and their interactions with the antibody are described in this paper. The smallest peptide recognized with a high affinity by antibody 164-2 contains 11 residues (273-283). This peptide is recognized by antibody 164-2 with an affinity (KD = 7.5 x 10(-9) M) very close to that of the native beta 2 subunit, suggesting a high structural similarity of the epitope inside the protein and in the isolated peptide. The corresponding sequence of beta 2 is located in a region protruding from the protein surface that contains a beta-turn as unique element of secondary structure in the crystallographic model. The absence of interaction between antibody 164-2 and the octapeptide lacking the three residues at the C-terminal end of peptide 11 suggests that the beta-turn is important in the recognition by the antibody. Kinetic studies were performed to find out whether or not the binding of the antibody to the peptide involves any conformational adaptation. The dissociation equilibrium constant (KD), the dissociation rate constant (koff) and the association rate constant (kon) were measured for eight peptide/antibody complexes. The values obtained were compatible with a one-step reaction, suggesting that no important conformational adaptation is involved in the formation of the peptide/antibody complexes. Furthermore, it has been shown that differences in affinity of antibody 164-2 for the various peptides were mainly due to differences in the dissociation rate constants (koff) and not in the association rate constants (kon). The exceptional location of the epitope in the native protein and the unusually high affinity of the 11-residue peptide for mAb 164-2, makes this peptide a good model for studying the interaction between an antibody and a continuous epitope of a protein.

Properties of monoclonal antibodies selected for probing the conformation of wild type and mutant forms of the P22 tailspike endorhamnosidase.

Eleven species of monoclonal antibodies directed against the trimeric P22 tailspike endorhamnosidase have been selected and characterized. Seven of these antibodies recognize the native tailspike, both isolated and assembled onto the virion, and prevent phage infection. Four antibodies react with denatured forms of the tailspike as well as with the plastic absorbed tailspike. Three of these latter prevent the tailspike from assembling onto the phage head. The antibodies have been tested against tailspike proteins carrying single amino acid substitutions at 15 different sites on the protein. Two of these mutations interfere with binding by a set of the monoclonals, indicating that they disrupt the epitopes for these antibodies. Since amino acid replacements corresponding to the temperature-sensitive folding mutations do not change the conformation of the native protein, these mutant proteins may be particularly useful for mapping epitopes. Amber fragments of the tailspike chain are recognized predominantly by the anti-denatured antibodies suggesting either that they are conformationally closer to folding intermediates than to the native tailspike or that the epitopes recognized by anti-native antibodies are carried by the C-terminal end of the native protein. Immunochemical detection by an anti-denatured antibody, after sucrose gradient sedimentation of a large 55-kDa amber fragment, indicates a monomeric rather than a trimeric state. This suggests that the missing C-terminal region is important for the trimerization reaction. Such N-terminal amber fragments may be useful models for studying with the monoclonal antibodies the nascent chain emerging from the ribosome.

Polypeptide-antibody binding mechanism: conformational adaptation investigated by equilibrium and kinetic analysis.

The mechanism of polypeptide-antibody binding was analysed by kinetic and equilibrium studies to find out whether or not the binding of an antibody to a large protein or a polypeptide antigen behaves as a one-step reaction or involves conformational adaptation. Three monoclonal antibodies recognizing 3 distinct epitopes on the C-terminal domain (F2) of Escherichia coli tryptophan synthase beta 2 subunit were used. The dissociation equilibrium constant (KD), the association rate constant (kon) and the dissociation rate constant (koff) of these antibodies for the native beta 2 subunit, its C-terminal fragment (F2) and different polypeptides obtained by chemical cleavage of the F2 fragment were measured. It was found that for some polypeptide-antibody complexes, binding could not be described as a one-step association-dissociation reaction, thus indicating the existence of conformational adaptation upon antibody-antigen complex formation. It was also shown that differences in affinities of a given antibody for its epitope carried by different polypeptides were mainly due to differences in the dissociation rate constant (koff) and not in the association rate constant (kon). Moreover, the immunoreactivity of various polypeptides obtained by cleavage of the F2 fragment enabled us to localize the 3 epitopes on the beta chain in light of the 3-dimensional structure of tryptophan synthase described recently by Hyde et al. (1988).

Epitope localization in antigen-monoclonal-antibody complexes by small-angle X-ray scattering. An approach to domain organization in the beta 2 subunit of Escherichia coli tryptophan synthase.

Each polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) is made of two domains, F1 (N-terminal) and F2 (C-terminal). To determine the relative position of these domains in the native protein, complexes between beta 2 and Fab fragments from two monoclonal antibodies, one specific for F1 (68-1) and the other for F2 (93-6), have been prepared and purified. Small-angle X-ray scattering measurements have been made on solutions of each complex. From the experimental scattering curves obtained, computer modeling leads to structural models of the two beta 2-Fab complexes. Though relatively low, the resolution of these models allows the localization on beta 2 of the antigenic sites recognized by the two antibodies, to show that the C-terminal F2 domains lie at the distal ends of the elongated beta 2 protein, and to show how steric hindrance prevents beta 2, though structurally and functionally dimeric, from binding more than one Fab 93-6 fragment per dimer.

Conformational changes induced by domain assembly within the beta 2 subunit of Escherichia coli tryptophan synthase analysed with monoclonal antibodies.

The effects of domain assembly on the conformation of the F1 (N-terminal) and F2 (C-terminal) domains of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were analysed using six monoclonal antibodies which recognize six different epitopes of the native beta 2 subunit (five carried by the F1 domain and one carried by the F2 domain). For this purpose, the affinity constant of each monoclonal antibody for the isolated domains F1 or F2, the associated domains in the trypsin-nicked apo-beta 2 and in the native apo-beta 2 subunits were determined, both with the intact immunoglobulin and the Fab fragment. It was found that the association of the F1 and F2 domains within beta 2 is accompanied by structural changes of the two domains, as detected by variations of their affinity constants for the monoclonal antibodies.