Prediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363-3731. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B-subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions. AntigenicityPrediction Antipeptide antibody Syntheticpeptide

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The complete primary and tertiary structure ofp-hydroxybenzoate hydroxylase is now known. The amino acid sequences of the two largest CNBr peptides have been fitted to the electron-density map at 0.25-nm resolution. The parts of the polypeptide chain contributing the residues to the FAD-binding site and the residues of the substratebinding site have been identified. The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure. Here the substrate, p-hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD.Many side chains from the C-terminal part of the polypeptide chain are involved in subunit-subunit interactions. In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.p-hydroxybenzoate hydroxylase from Pseudomonus ,fluerescens belongs to the class of flavin external monooxygenases. The enzyme catalyzes the incorporation of one atom from molecular oxygen in the substrate p-hydroxybenzoate with reduction by NADPH of the other oxygen atom. The enzyme forms dimers with two identical subunits. The monomer has a n~olecular weight of 43 x 1 O3 and contains no disulfide bridges [1,21.The three-dimensional structure of p-hydroxybenzoate hydroxylase with bound substrate has been determined at a resolution of 0.25 nm 131. In this complex the flavin is in the oxidized state and adopts a flat configuration. The entire FAD group is in an extended conformation and has numerous contacts with the protein. The polypeptide chain, which could be traced in the electron-density map, folds into three domains. Amino acids from all domains are involved in FAD binding. The substrate molecule is in close proximity to the isoalloxazine ring system. The enzyme contains much secondary structure, both a-helix (26%) and fi-sheet (16%). Moreover, the X-ray analysis confirms that the enzyme occurs as a dimer [3]. In the crystal the two subunits are related by a crystallographic twofold axis [3,4]. The contact area is formed by a domain predominantly built up from a-helices. The site of subunit interaction is well separated from the active site [3].In order to be able to interpret the electron-density map in terms of all the amino acid side chains, the primary structure of p-hydroxybenzoate hydroxylase was determined. The enzyme contains six methionines, one of which is N-terminal. After CNBr cleavage five peptides and free homoserine were isolated. The alignment of the CNBr fragments was deduced from the 0.25-nm electron-density map and sequence data. TheThe studies on peptide CB2 were part of the Ph. D. thesis of J. Hofsteenge presented in 1981 to the Rijksuniversiteit Groningen ; the studies on peptide CB1 will form part of the Ph. D. thesis of W. J. Weijer to be presented to the same university.Enzymes. p-Hydroxybenzoate hydroxylase (EC 1.14.13.2); glUtdthione reductdse (EC 1.6.4.2); D-amino-acid oxidase (EC 1.4.3.3); ferredoxin-NADP+ oxidoreductase (EC 1.1 8.1 2). __ -.-isolated f...

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