A series of peptides possessing the structure (L-Tyr-L-Ah-L-Glu)., with n = 1, 2, 3,4, 7, 9, and 13 was synthesized. The oligomers up to n = 4 were prepared by stepwise synthesis using the N-hydroxysuccinimide ester of the tripeptide derivative for the elongation of the chain. The oligomers with n = 7 , 9 , 13 were prepared by a polycondensation technique, followed by gel filtration.The circular dichroism spectra of the above oligopeptides were measured in the wavelength range 200-330 nm in a solution of 0.15 M sodium chloride -0.02 M sodium phosphate, pH 7.4, conditions a t which the high molecular weight polytripeptide (Tp-Ala-Gh), exists in a helical conformation. The circular dichroic spectrum of the high molecular weight polymer exhibits two negative ellipticity bands: an intense band a t 220 nm ( . The other oligopeptides (n = 2 to 13) exhibit positive bands in the 227 nm region, with ellipticity values that decrease as the degree of polymerization increases. Except for (Tyr-Ala-Glu),,, which has a negative peak centered a t 216 nm, circular dichroic spectra of the other oligomers (n = 1 to 9) in the 200-210 nm region resemble that of random coils. I n the 260-290 nm region, the circular dichroic curves of the oligopeptides gradually approach the 272 nm band of the polypeptide (Tyr-Ala-Glu),, as the degree of polymerization increases. Under physiological conditions, only the (Tyr-Ala-Glu),, shows an indication of helical content.The high molecular weight polytripeptide with the repeating sequence L-tyrosyl-L-alanyl-L-glutamyl was synthesized and characterized by Ramachandran et al. [l-31. This polymer, which has been shown to possess an &-helical conformation under physiological conditions, was used by us for an investigation of the nature of antigenic determinants in macromolecules [3-61. I n connection with this study, which is described in the accompanying paper [4] and others to follow [5,6], we have synthesized a series of peptides of the structure (Tp-Ala-Glu),, where n = 1 , 2 , 3 , 4, 7, 9, 13. The peptides (Tyr-Ala-Glu),, with n = 1 to 4 were prepared by a stepwise synthesis using the succinimide ester of N,O-dibenzyloxycarbonyl-Ltyrosyl-L-alanyl-y-benzyl-L-glutamate as a unit of elongation. After removal of the blocking groups the free peptides were fractionated by means of gel filtration, resulting in essentially homogeneous products. The peptides (Tp-Ala-Glu), , with n = 7, Unusual Abbreviations. Oligopeptides (Tyr-Ale-Glu),, peptides containing up to 13 residues each of L-tyrosine, L-alanine and L-glutamic acid in regular alternating sequence ; polypeptide (Tyr-Ala-Gh& a helical polymer of the regular alternating tripeptide Tyr-Ala-Glu with n approx. 200; polypeptide (Tyr,Ala,Glu)n, random copolymer of L-tyrosine, L-alanine and L-glutamic acid. 21 Eur.
The high molecular weight copolymer (mol.wt. = 75000) containing the repeating sequence-(L-Tyr-L-Ala-L-G1u)-and denoted as polypeptide (Tyr-Ala-Gh), has previously been shown to possess an a-helical structure under physiological conditions. Studies were performed t o check whether monovalent fragments of antibodies derived from anti-(Tyr-Ala-Glu), antibodies could convert the partially helical oligopeptide (Tyr-Ala-Glu),, into a more helical conformation.Circular dichroic spectra of mixtures of the monovalent antibody fragments with (Tyr-AlaGlu),, were measured a t two different concentrations of both components. Analogous experiments were done with the same fragments and the polypeptide (Tyr-Ah-Glu),. When anti-(Tyr-AlaGlu), antibody fragments were mixed with (Tyr-Ala-Glu)13, there was a large difference spectrum centered at 278 nm, which is the ellipticity band of (Tyr-Ala-Glu),,. When the same fragments were mixed with the polypeptide (Tyr-Ah-Glu),, a difference spectrum was observed at the ellipticity band of the polypeptide centered at 275 nm. It was concluded that anti-(Tyr-Ala-Glu), monovalent antibody fragments could not only convert the (Tyr-Ala-Glu),, into a structure more similar to the polypeptide (Tyr-Ah-Glu),, but could also stabilize the helical content of this polypeptide.Interaction between two macromolecules may induce conformational changes in both of them. Thus, substrates may bring about an "induced fit" with enzyme molecules [I]. The immunochemical reaction between an antigen and an antibody may similarly result in changes both within the areas including antibody combining sites and the areas including antigenic determinants. In the case of the antibody, recent studies have suggested that a monovalent hapten causes no conformational change in the antibody molecule as detected by circular dichroic measurements [2] and by differential sedimentation [3], whereas interaction with a divalent hapten causes a slight change in frictional ratio of the antibody molecule [3].Several reports have appeared in recent years describing changes in the conformation of the antigenic molecule upon reaction with antibodies. Thus, myoglobin is converted into apomyoglobin upon reaction with anti-apomyoglobin antibodies [4], whereas enzymically inactive /3-galactosidase mutants gain activity upon reaction with antibodies to the wild type enzyme [ 5 ] .I n connection with an investigation of the role of macromolecular conformation in immunogenicity and antigenic specificity [6,7], we have described in the preceding paper [8] antibodies against conformation-dependent determinants of an &-helical copolymer of the ordered periodic sequence (Tyr-AlaGlu),. The ability of these antibodies to react with oligopeptides containing the repeating sequence L-tyrosyl-L-alanyl-L-glutamyl [8,9] as followed by their capacity to inhibit the homologous precipitin system, increased with increasing length of the oligopeptides. Thus, the tripeptide Tyr-Ala-Glu did not inhibit the precipitin reaction, (Tyr-AlaGlu), inhibited poorly, whe...
Two synthetic copolymers have been used in order to investigate the role of conformation in immunogenicity and antigenic specificity. The first one is a high molecular weight ordered copolymer composed of the repeating sequence L-tyrosyl-L-alanyl-L-glutamyl. This polymer exists as an n-helix under physiological conditions. I n the second polymer, the tripeptide L-tyrosyl-Lalanyl-L-glutamic acid is attached to branched poly-m-alanine. I n this branched polymer the tripeptide exists as a random coil. These polymers elicit the formation of antibodies with distinct specificities, since there was no cross-precipitation reaction between the two systems. Furthermore, antibodies against the branched polymer were efficiently inhibited by the tripeptide L-tyrosyl-L-alanyl-L-glutamic acid, whereas antibodies against the helical polymer were inhibited neither by the tripeptide, nor by other small peptides of sequences related to the primary structure of the helical polymer. However, inhibition was obtained with oligopeptides possessing the general formula (Tyr-Ala-Glu), (with n = 3,4, 7, and 9), the inhibitory capacity increasing with the value of n. Peptides with n values of 7 and 9 were efficient inhibitors, whereas (Tyr-Ale-GIU),~ precipitated with the antibodies. The dependence of inhibitory capacity on n suggests that the peptides with n = 2-9 undergo transconformation (from random coil to a-helix) upon reaction with the antibody combining site.The immunological studies showed that the antigenic determinants of the n-helical polymer are conformation-dependent, whereas the branched polymer contains sequential determinants.
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