8536d_ch03_057-075 8/7/02 9: 18 AM Page 66 mac79 Mac 79: 45_BW: Goasby et al./ Immunology 5e PART II Generation of B-Cell and T-Cell Response native protein conformation for their topographical struc- these epitopes are conformational determinants dependent ture. One well-characterized nonsequential epitope in hen on the overall structure of the protein. If the intrachain disul egg-white lysozyme(HEL) is shown in Figure 3-6b Although fide bonds of HEL are reduced with mercaptoethanol, the the amino acid residues that compose this epitope of HEL are nonsequential epitopes are lost; for this reason, antibody to far apart in the primary amino acid sequence, they are native hel does not bind to reduced hEL. brought together by the tertiary folding of the protei The inhibition experiment shown in Figure 3-7 nicely Sequential and nonsequential epitopes generally behave demonstrates this point. An antibody to a conformational differently when a protein is denatured, fragmented, or re- determinant, in this example a peptide loop present in native duced. For example, appropriate fragmentation of sperm HEL, was able to bind the epitope only if the disulfide bond whale myoglobin can yield five fragments, each retaining one that maintains the structure of the loop was intact. Infor- quential epitope, as demonstrated by the observation that mation about the structural requirements of the antibody antibody can bind to each fragment On the other hand, frag- combining site was obtained by examining the ability of mentation of a protein or reduction of its disulfide bonds of- structural relatives of the natural antigen to bind to that an- ten destroys nonsequential epitopes. For example, HEL has tibody. If a structural relative has the critical epitopes present four intrachain disulfide bonds, which determine the final in the natural antigen, it will bind to the antibody combining protein conformation(Figure 3-7a). Many antibodies to site, thereby blocking its occupation by the natural antigen. HEL recognize several epitopes, and each of eight different In this inhibition assay, the ability of the closed loop to in epitopes have been recognized by a distinct antibody. Most of hibit binding showed that the closed loop was sufficiently (a) Hen egg-white lysosome (b) Synthetic loop peptides 夺HN OOH 64-0-COOH H2N-oodpdoooooodpP ○ (c) Inhibition of reaction between HEL FIGURE3-7Experimental demonstration that binding of antibody to conformational determinants in hen egg-white lysozyme(HEL) depends on maintenance of the tertiary structure of the epitopes by intrachain disulfide bonds. (a) Diagram of HEL primary structure, in which circles represent amino acid residues. The loop(blue circles) 60 formed by the disulfide bond between the cysteine residues at posi- tions 64 and 80 constitutes one of the conformational determinants in HEL.(b) Synthetic open-loop and closed-loop peptides corre- ponding to the HEL loop epitope.(c) Inhibition of binding between HEL loop epitope and anti-loop antiserum. Anti-loop antiserum was a Closed synthetic loop first incubated with the natural loop sequence, the synthetic closed loop peptide, or the synthetic open-loop peptide; the ability of the an- tiserum to bind the natural loop sequence then was measured. The absence of any inhibition by the open-loop peptide indicates that it does not bind to the anti-loop antiserum (Adapted from D. Benjam et al, 1984, Annu. Rev. Immunol. 2: 67 1 Ratio of loop inhibitor to anti-loop antiserumnative protein conformation for their topographical struc￾ture. One well-characterized nonsequential epitope in hen egg-white lysozyme (HEL) is shown in Figure 3-6b. Although the amino acid residues that compose this epitope of HEL are far apart in the primary amino acid sequence, they are brought together by the tertiary folding of the protein. Sequential and nonsequential epitopes generally behave differently when a protein is denatured, fragmented, or re￾duced. For example, appropriate fragmentation of sperm whale myoglobin can yield five fragments, each retaining one sequential epitope, as demonstrated by the observation that antibody can bind to each fragment. On the other hand, frag￾mentation of a protein or reduction of its disulfide bonds of￾ten destroys nonsequential epitopes. For example, HEL has four intrachain disulfide bonds, which determine the final protein conformation (Figure 3-7a). Many antibodies to HEL recognize several epitopes, and each of eight different epitopes have been recognized by a distinct antibody. Most of these epitopes are conformational determinants dependent on the overall structure of the protein. If the intrachain disul￾fide bonds of HEL are reduced with mercaptoethanol, the nonsequential epitopes are lost; for this reason, antibody to native HEL does not bind to reduced HEL. The inhibition experiment shown in Figure 3-7 nicely demonstrates this point. An antibody to a conformational determinant, in this example a peptide loop present in native HEL, was able to bind the epitope only if the disulfide bond that maintains the structure of the loop was intact. Infor￾mation about the structural requirements of the antibody combining site was obtained by examining the ability of structural relatives of the natural antigen to bind to that an￾tibody. If a structural relative has the critical epitopes present in the natural antigen, it will bind to the antibody combining site, thereby blocking its occupation by the natural antigen. In this inhibition assay, the ability of the closed loop to in￾hibit binding showed that the closed loop was sufficiently 66 PART II Generation of B-Cell and T-Cell Responses FIGURE 3-7 Experimental demonstration that binding of antibody to conformational determinants in hen egg-white lysozyme (HEL) depends on maintenance of the tertiary structure of the epitopes by intrachain disulfide bonds. (a) Diagram of HEL primary structure, in which circles represent amino acid residues. The loop (blue circles) formed by the disulfide bond between the cysteine residues at posi￾tions 64 and 80 constitutes one of the conformational determinants in HEL. (b) Synthetic open-loop and closed-loop peptides corre￾sponding to the HEL loop epitope. (c) Inhibition of binding between HEL loop epitope and anti-loop antiserum. Anti-loop antiserum was first incubated with the natural loop sequence, the synthetic closed￾loop peptide, or the synthetic open-loop peptide; the ability of the an￾tiserum to bind the natural loop sequence then was measured. The absence of any inhibition by the open-loop peptide indicates that it does not bind to the anti-loop antiserum. [Adapted from D. Benjamin et al., 1984, Annu. Rev. Immunol. 2:67.] (b) Synthetic loop peptides CYS 80 64 CYS Open loop Closed loop CYS 80 CYS 64 COOH H2N (a) Hen egg–white lysosome Disulfide bond COOH H2N 64 80 16 Ratio of loop inhibitor to anti–loop antiserum 100 Inhibition, % 0 8 80 60 40 20 Natural loop Closed synthetic loop Open synthetic loop (c) Inhibition of reaction between HEL loop and anti–loop antiserum 8536d_ch03_057-075 8/7/02 9:18 AM Page 66 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: