! \j 4 Department of Biology A-- Johns Hopkins University Baltimore, Maryland Much of the current research in the field of synthetic vaccines is based-on the assumption that fragments of antigens --for example, fragments oyviral coat proteins or toxlnsc can, upon attachment to a neutral carrier structure tion into patients, induce the synthesis of antibodies that will recognize and immobilize the pathogenic agent. Anti- bodies recognize native conformations or protein determinants ("conformational determinants") and also primarily random 'peTtide fragments ("sequential determinants") (1). It becomes important to determine the extent to whic'fi the latter, random structures can take on the "native format" that such fragments exhibit when part of the native protein structure. such involvement in an equilibrium between the ran-w proper-&nation woula tne fragment serve as a mean ng . Va+ne pr n m rsTr@o (z) we snofi that a fragment of hen's egg white lysozyme could be synthesized, attached to a carrier module, and be injected into animals with the produc- tion of antibody that would react with, and inactivate the original enzyme molecule. The synthetic material was, in a sense, a "vaccine" against lysozyme. As will be seen in Fig- ure 1, this antigenic lysozyme "loop" was stabilized in a suitable conformation by a disulfide bridge. Peptide frag- ments without such stabilization would tend to occupy a large set of random conformations, very few of which would be likely to be recognized by an antibody formed against the native, parent protein molecule. We have used affinity chromatography as a tool in the study of the interaction of peptides with antibodies (Anti- bodyW) raised against the native protein. By the attachment 356 CHRISTIAN B. ANFINSEN ET AL. ASN PESIN ALA SER CYS PRO ILE ASN ALA LEU c tz om se2 Cltovoqt from rt*an and rtmovol of blockmq qfoups by onhydrous HF DEBLOCKED PEPTIDE Rtducllon by mtrcovlotlhonol and rtorldohon I" 08, SYNTHETIC LOOP FIGURE 1. The solid phase synthesis, deprotection and ring closure of a peptide "loop" in hen's egg white lysozyme. of fragments of the protein chain to a Sepharose supporting backbone, subfractions of the mixed Antibody population can be isolated. The isolation of such monospec!!fic antibodies provides reagents that are no longer-g. Thus, one can deal quantitatively with antigen-antibody reactions in solution, and measurements are made more easily and more accu- rately. The binding to such columns of monospecific antibod- ies already suggests that fragments of the protein molecule, which appear to be totally lacking in structure as determined by the usual physical measurements, do indeed occupy ordered structure for at least a small portion of their lifetime. Thus, a peptide appears to "flicker" between the random form and the form that the peptide occupies when it is part of A r. the PROTEIN FOLDING 357 FIGURE 2. The amino acid sequence of staphylococcal nuclease. FIGURE 3. Drawing of the three-dimensional structure of staphylococcal nuclease. 358 CHRISTIAN B. ANFlNSEN ET AL FIGURE 4. Some fragments prepared from staphylococcal nuclease. Top : location of pleated sheet and helical sec- tions of nuclease. Fragments labelled 6-48 and 49-149 are produced by limited trypsin digestion. Peptide l-126 (and the fragment 127-149, not shown) are obtained by trypsin digestion of trifluoroacetylated nuclease with subsequent removal of the TFA protecting groups. Peptide 99-149 is one of five fragments produced by cyanogen bromide digestion of the protein. native protein structure. Guided by its amino acid sequence, it can occasionally assume its conformation in the intact parent molecule. Much of this work has been done with the convenient pro- tein, staphylococcal nuclease (Fig. 2 and 3), which is devoid of SS bridges and undergoes very rapid denaturation and rena- turation. It may be cleaved into a variety of peptide frag- ments by enzymic or chemical methods (Fig. 4). The purified was fractionated by sequential immunoabsorption and elution using columns of Sepharose to which one or another of the peptide fragments had been attached. The preparation of ne non-precipitating, but inactivating antibody, is summar- in Figure 5 (3). i.e., anti-native was first isolated on a then successively fraction- contained antibody directed against This fraction was monodeterminant, and although it still combined the 99-126 portion of native nuclease. When anti-(99-126) was added to nuclease, the enzyme was inactivated (3,4). Hogever, addition of increasing amounts PROTEIN FOLDING ANTI- NUCLEASE ANTIBODIES 20- A~II-NucI~~~~ QI %ph0~-(l-l49) 0 25 Ani!-Nucleose cm Sechmse-l99-t491 359 lot Antd39-149)" / ot A;@27-`4g1n -; +I , 72, , ,22-y-72,- 0 IO 20 30 40 50 FIGURE 5. Fractionation of antibody against native nuclease by sequential absorption on Sepharose columns to which either nuclease, peptide 99-149 or peptide 127-149 have been attached. of the randomly structured peptide 99-149 led to increasing release of the antibody-bound enzyme. Determinations of the successive activity levels permitted calculation of the equi- librium constant relating random structure, and structure sufficiently similar to the native "format" of the peptide to permit competition with nuclease for antibody (99-126)*. for assuming their native format. For example, peptide (127- 149) and its monospecific antibodyN were employed in an analy- sis similar to the above study of competition between intact nuclease and peptide fragment, and yielded the same estimate of folding frequency. Using similar methods, one can determine the frequency of unfolding of a native structure such as nuclease (5). How CHRISTIAN B. ANFINSEN ET AL. COMPETITION ASSAY ,149) _ AbR.14 C-(99-149) I AbR'NR + Rabbit dnti-Goat Imunoglobulin 14C-(99-149), NR, NN precipitate: AbR, AbR* 14 C-(99-149), AbR'NR FIGURE 6. Double antibody procedure for establishing the degree to which native nuclease can unfold into a family of denatured forms. often is a protein molecule sufficiently unwound to be recog- nized by an antibody directed against a random polypeptide structure? For these studies, the peptide (99-149). This ant f%%t purified from the IgG fraction by The purified antibody was then reacted It was found that nuclease could? mpete with a it for tn extent consistent This indicates par- tial unfolding of nuclease. d in Figure 6, the extent of competition could be measured by using rabbit anti- goat immunoglobulin to determine the fraction of the labelled peptide in the precipitate and supernatant components. When the experiments were carried out at &and 30 C, the confor- mational equilibrium constants were 4000 and &O, respectively. Addition of stabilizing ligands for nuclease (thymidine di- phosphate and=cium ionsT J&d to a constant of 40,000. The tendency for certain peptide frmein molecules to occupy their native "format" in solution may ecome a s for the p'reparation or a usetul vaccine. Such an approach has been taken%y Arnon ana Se-colleagues (6) at the Weizmann Institute of Science and, more recently, by Lerner and the synthetic vaccine group at the Scripps Clinic of the University of California in San Diego (7). PROTElN FOLDING REFERENCES 361 1. 2. 3. 4. 5. 6. 7. Sela, M., Schechter, A., Schechter, I., and Borek, F., Cold Spring Harbor Symp. Quant. Biol. 32, 537 (1967). Arnon, R., Maron, E., Sela, M., and Anfinsen, C. B., Proc. Natl. Acad. Sci. U.S.A. 68, 1450 (1971). Sachs, D. H., Schechter, A. N., Eastlake, A., and Anfin- sen, C. B., J. Immunol. 109, 1300 (1972). Sachs, D. H., Schechter, A., Eastlake, A., and Anfinsen, C. B., Proc. Natl. Acad. Sci. U.S.A. 69, 3790 (1972). Furie, B., Schechter, A. N., Sachs, D. H., and Anfinsen, C. B., J. Mol. Biol. 92, 497 (1975). Arnon, R., Sela, M., Parant, M., and Chedid, L., PTOC. Natl. Acad. Sci. U.S.A. 77, 6769 (1980). This volume.