No abstract

The contact interactions between a synthetic peptide and three different anti-peptide monoclonal antibodies have been studied by nuclear magnetic resonance (NMR). The synthetic peptide is CTP3 (residues 50-64 of the B subunit of cholera toxin) suggested as a possible epitope for synthetic vaccine against cholera. The hybridoma cell lines TE33 and TE32 derived after immunization with CTP3 produce antibodies cross-reactive with the native toxin. The cell line TE34 produces anti-CTP3 antibodies that do not bind the toxin. Selective deuteriation of the antibodies has been used to simplify the proton NMR spectra and to assign resonances to specific types of amino acids. The difference spectra between the proton NMR spectrum of the peptide-Fab complex and that of Fab indicate that the combining site structures of TE32 and TE33 are very similar but differ considerably from the combining site structure of TE34. By magnetization transfer experiments with selectively deuteriated Fab fragment of the antibody, we have found that in TE32 and TE33 the histidine residue of the peptide is buried in a hydrophobic pocket of the antibody combining site, formed by a tryptophan and two tyrosine residues. The hydrophobic nature of the pocket is further demonstrated by the lack of any pH titration effect on the chemical shift of the C4H of the bound peptide histidine. In contrast, for TE34 we have found only one tyrosine residue in contact with the histidine of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specificities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.

The binding of fluorescently labeled heavy meromyosin (HMM) and heavy meromyosin subfragment-1 (S-1) to thin filaments of myofibrils and of rabbit psoas muscle fibers was measured under conditions of rigor and contraction. The fragments diffused rapidly into the myofibrillar space and bound specifically to the thin filaments. The fragments bound strongest and in a uniform fashion to myofibrils in which the competition from indigenous myosin was abolished by removing it with Hasselbach-Schneider solution. Under these conditions, the rigor Ka values for HMM and S-1 were 1.5 x 10(6) M-1 and 4.8 x 10(4) M-1, respectively. The stoichiometry of binding was measured by independently estimating the concentration of actin sites. S-1 was found to be capable of saturating all available actin sites in a myofibril or a fiber, but HMM could only occupy 50% of the sites.

Tryptic digestion of myofibrils was used to assess the interaction of crossbridges with thin filaments in the presence of ATP analogues. The relative amounts of 200 kDa fragment produced by trypsin from myosin heavy chain when the crossbridge is attached to actin, and of 160 kDa fragment produced when the crossbridge is detached from actin, served as a measure of crossbridge-actin interaction. In rigor only the 200 kDa fragment was produced suggesting that a great majority of the crossbridges were strongly attached to actin; in the presence of MgPPi at 0 degrees C only the 160 kDa fragment was finally produced suggesting that eventually all crossbridges detached from actin. In the presence of MgPPi or MgAMPPNP at 25 degrees C both 200 and 160 kDa fragments were present for several minutes after myosin heavy chain had been completely digested, suggesting that two populations of crossbridges (attached and detached) co-existed at the same time within the myofibril. It is concluded that the addition of ATP analogues to muscle does not simply affect the chemical equilibrium of binding of myosin heads to actin but that it causes rapid dissociation of one crossbridge population without significant effect on binding to actin of the remaining crossbridge population.