Reprinted from the 27th Symposium of the Society for Developmental Biology DEVELOPMENTAL Biotocy SuPPLEMENT 2, 1968 Copyright © 1968 by Academic Press Inc. Printed in U. S. A. DEVELOPMENTAL BIOLOGY SUPPLEMENT 2, 1-20 (1968) I. SELF-ASSEMBLY OF MACROMOLECULAR STRUCTURES Spontaneous Formation of the Three-Dimensional Structure of Proteins Curist1an B. ANFINSEN Laboratory of Chemical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland INTRODUCTION Our major consideration in this symposium will be the emergence of order during cellular differentiation and growth. The concept “emerging order” implies an organized, genetically complex process taking place over a reasonably extended stretch of time. In contrast, the restatement of linear genetic information in the form of three- dimensional protein structure results from a rapid and spontaneous interaction of amino acid side chains with each other, with the com- pleted polypeptide backbone, and with the environment, without the necessity for additional genetic information (Anfinsen, 1967; Epstein et al., 1963). The achievement of this unique geometry might be visualized as a rather helter-skelter process. An almost infinite number of sets of interactions are possible as an extended polypeptide chain coils upon itself (Fig. 1). If the process of folding involved even a small fraction of this number of conformational states, the specific folding of the chain could clearly require considerable time. It is prob- able that the rapidity of folding is made possible through the forma- tion of one or more “nucleation sites” by side chain interactions that would predispose, during subsequent interactions, to the tertiary struc- tural characteristics of the native structure. The only obvious driving force during this approach to native conformation is the selection of progressively more stable conformations with ultimate fixation of geom- etry in the form possessing the most favorable free energy of conforma- tion, the native protein. Thus, unlike the complex predetermined pattern of successive changes occurring during differentiation, the cell must rely, in its first steps of development, on a relatively random 1 © 1968 by Academic Press Inc. 2 CHRISTIAN B. ANFINSEN process but involving explicit information—the amino acid sequence of a polypeptide chain. It has been suggested (Phillips, 1967) as an alternative mechanism that a polypeptide chain may progressively assume a three-dimensional conformation similar or identical to that which it occupies in the com- pleted protein molecule, as synthesis proceeds from the NH.-terminus toward the COOH-terminal end of the chain. However, the weight of evidence available at the present time, some of which I shall mention Polypeptide chain Native protein Fic. 1. Schematic drawing showing the conversion of an extended polypeptide chain to a native protein. During this oxidative process, sulfhydryl groups are paired to form disulfide bonds, and amino acid residues, widely separated in a linear sense, are brought into spatial proximity to form an active center. below, appears to be consistent with a process in which tertiary struc- ture appears only upon completion of translation of the genetic quan- tum of information. With the exception of the synthesis of certain RNA molecules, the information in a chain is expressed in a form useful to a cell as linear “bursts” of polypeptide chains, Each chain represents the raw material for a function that is performed by the corresponding protein molecule. Evolution in its simplest form has consisted of the continuous selection FOLDING OF PROTEINS 3 of organisms on the basis of the adequacy of the summation of their proteins to constitute a cell system favorable to self-reproduction under the current ecological situation. The sequences of the polypep- tide chains that are synthesized are so constituted that they assume, in a spontaneous manner, unique geometric shapes that are endowed with the function in question. Most of our information has come from a study of proteins that contain disulfide bonds as cross-links and the reversibility of refolding has been tested by examining the reformation of correct pairs of half-cystine residues, together with the restoration of biological ac- tivity and various physicochemical properties. The statistics of the situation are shown in Table 1, which lists the number of possible ways in which a given number of half-cystine residues can combine TABLE 1 Toe NUMBER oF Ways IN Wincn 22 SULFHYDRYL Groups Can ComBINE to Form 7 DisuLtripg Bonxps Number of bonds Number of combinations 1 1 2 3 3 15 4 105 5 945 6 10895 Np, = (2a)! 7 135135 2i(2n — 2) Yj! s 2027025 9 34459425 10 654729075 11 137493 L0575 12 316234143225 13 7905853580625 14 213458046676875 15 6190283353629375 16 191898783962510625 17 6332859870762850625 18 22164309547669977 1875 19 $2200794532637891559375 20 31983098677 2877770815625 21 131113070457687988603440625 22 563862029680583509947946875 23 2537379 1335626257 947657609375 24 1192568 192774434 123539907640625 25 5843584 1445947272053455474390625 4 CHRISTIAN B, ANFINSEN to form SS bonds upon oxidation. These numbers show, for example, that in the case of the y-globulin molecules, the random chance ot forming the correct 23 SS bonds from the available 46 half-cystine residues is 1 in 2 x 10°*. In the case of pancreatic ribonuclease, which contains 8 half-cystine residues, 105 possible sets of 4 SS bonds can be made, only one of which is the native structure. Since much of the evidence for the spontaneity and uniqueness of polypeptide folding has been summarized earlier, I shall present here only a schematic picture. Figure 2 depicts the renaturation of what we have called a Fic. 2. The spontaneous conversion of a randomly crosslinked protein deriva- tive to the native form under conditions favoring disulfide interchange. Structural regions of the molecule that are involved in the active center are indicated by crosshatching. “scrambled” ribonuclease molecule. After complete reduction of the 4 disulfide bonds in the native protein, the reduced random chain was allowed to reoxidize under conditions leading to a random mixture of disulfide bonds (Haber and Anfinsen, 1962), shown diagrammatically in the upper portion of the figure. The thermodynamic instability of this scrambled mixture is demonstrated by the observation that expo- sure to conditions favoring disulfide interchange induced rapid rear- rangement of the disulfide bonds with the formation in almost quanti- tative yields of the native enzyme with its correct SS pairs. By using as a catalyst for the interchange process an enzyme from microsomal membranes that we have recently isolated, the renaturation process can be made to occur in vitro (Fuchs et al., 1967) at a rate which is quite consistent with the estimated length of time required for the synthesis of a ribonuclease molecule in vivo, namely about 2 minutes (Dintzis, 1961; Canfield and Anfinsen, 1963). This experimental result militates against the concept of obligatory progressive folding during FOLDING OF PROTEINS 5 the NH.-terminal to COOH-terminal synthesis of the chain since the scrambled collection of isomers is devoid of the features of tertiary structure that one finds in the native enzyme. We have recently carried out some pertinent experiments on the thermodynamic stability of the RNase derivative, RNase-S (Kato and Anfinsen, unpublished results). This material, prepared by the con- trolled cleavage of a single bond between residues 20 and 21 in bovine pancreatic ribonuclease by the enzyme, subtilisin, may be separated into its two noncovalently bonded components, RNase-S-protein and RNase-S-peptide (Richards and Vithayathil, 1959). The former, con- taining all the four disulfide bonds of the native protein, is inactive without the addition of the peptide moiety. To test whether the S-protein portion contained sufficient information to determine the specific folding that would lead to proper pairing of the eight half- cystine residues, samples were subjected to conditions of disulfide interchange under catalysis by the rearranging enzyme from micro- somes mentioned above. This enzyme, after prereduction of its single essential SH group, will catalyze disulfide rearrangement without need for added mercaptoethanol or other SH reagent. As summarized in Fig. 3, addition of the enzyme to S-protein solutions caused rapid loss of the capacity of the S-protein to be activated by addition of 1.3 equivalents of S-peptide. Peptide maps of pepsin digests indicated the presence of random SS pairing. [The residual activity may represent material which does not contain all the normal four SS bonds of ribo- nuclease. The recent observations of Neumann e¢ al. (1967) on the preparation of a fully active derivative of RNase containing only two intact disulfide bonds indicate that two of the native disulfide linkages in this protein are superfluous from the standpoint of in vitro activity. Consistent with this view is the observation that fully reduced S- protein, when allowed to oxidize in the absence of S-peptide, with complete conversion of its 8 SH groups to 4 SS bonds, yields low levels of active material (Kato, unpublished; Haber and Anfinsen, 1961).] Upon addition of S-peptide to the largely inactivated S-protein solu- tion, the bulk of the activity was regenerated. Similar conclusions may be drawn from parallel experiments in which the formation of intermolecular, disulfide bonded aggregates of S-protein was studied in the presence and absence of S-peptide (Fig. 4) by turbidity measurements. Once again, the information contained in the S-peptide portion of RNase-S was required to deter- 6 CHRISTIAN B, ANFINSEN mine the native structure which, by inference, must represent the most thermodynamically stable form. Experiments similar to those I have just described for ribonuclease and S-protein have been carried out on a wide variety of protein molecules, both large and small, and the phenomenon appears to be a general one (Anfinsen, 1967). Perhaps the most dramatic example is 100 p--- poe 1 > 80 | 40 ~ PERCENT OF ACTIVITY 20}- — O . i woe wee 4 0 100 200 300 TIME, MINUTES Fic. 3. Inactivation and disulfide interchange of native RNase-S-protein cata- lyzed by prereduced interchange enzyme (I. Kato and C. B. Anfinsen, unpub- lished results; Fuchs et al., 1967). The arrow indicates the time of addition of RNase-S-peptide (1.3 equivalents relative to S-protein) to the reaction mixture. RNase-S-peptide (1.3 equivalents) was added to aliquots taken prior to the time marked by the arrow, and the mixtures were assayed for RNase activity. given by recent studies by Freedman and Sela (1966) on y-globulins. Both Haber (1964) and Whitney and Tanford (1965) showed that the (Fab), fragment of 7S ye antibodies, produced by papain digestion, could be subjected to full reduction of SS bonds with subsequent restoration of significant levels of specific antibody activity upon reoxidation. Freedman and Sela were able to repeat such experiments using undegraded, native antibody molecules by the trick of massive polyalanylation of the «amino groups of the purified rabbit-antibovine serum albumin. The addition of pi-polyalanyl side chains on proteins and polypeptides has been shown, in several instances, to confer much FOLDING OF PROTEINS 7 greater solubility on the products than that shown by the unpep- tidylated material. The 23 disulfide bonds of the protein (whose immunological activity was unchanged by the peptidylation) could then be reduced without formation of the otherwise insoluble, reduced heavy chain, a product of reduction that had been avoided by use ot papain fragments in the earlier experiments. The reduced forms of the soluble, polyalanylated light and heavy chains were reoxidized sepa- rately and finally recombined through oxidative formation of the [vi ees oe Sy. a E ° ime Ww & 1.0]- > oO 2 a oO © 3° w a y-Asn- Pro-Tyvr-Val-Pre-Val-His-Phe-Asp-Ala-Ser-VYal Fic, 6. A comparison of the amino acid sequences of rat (above) and bovine (below) pancreatic ribonucleases. The enclosed area contains the regions of identical sequence (Beintema and Gruber, 1967; Wyckoff, 1968). context of the bovine geometry, differences in sequences in the rat protein, often occurring in pairs and frequently far separated on the chain, make good sense in terms of structural stabilization. Many of these double replacements appear to permit the retention of interac- tion between neighboring lengths of the polypeptide chain that form stabilized, structural features of the three-dimensional model. For example, the substitutions of arginine and glutamic acid at positions 80 and 103, replacing the neutral serine-asparagine interaction in the bovine enzyme, may help maintain the stability of a loop in the struc- ture, but now by an electrostatic interaction. Other replacements lead to a conservation polarity or specific net charge in certain areas of the surface. Thus, replacement of the hydrophobically interacting methio- 12 CHRISTIAN B. ANFINSEN nine residue 79 in the bovine enzyme with leucine in the rat, involves little change in volume but a definite change in shape. Since the former residue is partly exposed in a pit in the bottom of the three- dimensional model, the change in shape can be accommodated and actually makes room for the extra volume of isoleucine 57 which replaces valine 57 in the bovine protein. Some of the double changes are less understandable when consid- ered in the context of other experimental data. The pair of conforma- tionally neighboring residues, Lys-61 and Gln-74, in the bovine enzyme became Gly and Lys, respectively, in the rat protein. Local charge is preserved by this set of replacements, but an examination of the three- dimensional model does not suggest any more subtle reason for “con- servatism,”’ such as preservation of a stabilizing interaction or the avoidance of a “hole” in the structure. Nevertheless, our studies on polyalanylated RNase, referred to above, show clearly that the «amino group of lysine-61 may be modified by the addition of a chain of 5-8 alanyl residues without interference with either activity or the capacity of the fully reduced polyalanyl-RNase to refold correctly after com- plete reduction and denaturation. Such a modification, although pre- serving net charge, moves the ionized amino group about 20A from the position of the original «amino group. Intracellular requirements of a more complex nature must underlie the genetic changes that lead to double replacements of this sort; it is clear that we have much to learn about the “design” of proteins in relation to function. EFFECTS OF INTERRUPTION OR MODIFICATION OF GENETIC INFORMATION Since function is a consequence of precise geometry, spontaneous and correct folding of a polypeptide chain might not occur after tam- pering with the integrity of the translated genetic information. It is of interest, therefore, to examine the adequacy of the information for folding in multichained proteins after various limited cleavages. Multichained proteins may be classified as follows: 1. Naturally occurring proteins containing more than one chain resulting from specific in vivo cleavage; this group includes, to my knowledge, only two examples—chymotrypsin and insulin. 2. Biologically active multichained molecules derived from single- chained proteins, produced by deliberate experimental cleavage of peptide bonds by protease treatment. This group of man-made deriva- FOLDING OF PROTEINS 13 tives is very small; RNase-S (Richards and Vithayathil, 1959), RNase-E (Klee, 1965), RNase-T (Ooi et al., 1963) (Fig. 7), and nuclease-T, -S, and -C (see Figs. 8 and 9). 3. Naturally occurring multichained proteins formed by disulfide bonding of two or more separately synthesized chains—the immuno- logically active globulins. 4. Oligomeric proteins, made up of noncovalently aggregated single chains. This very large group includes a variety of intracellular pro- teins whose multimeric structures permit “allosteric” modifications due to ligand interaction. subtilisin or Ribonuclease ———> RNase-S or R Nase-k elastase (124 residues) Fragment 1 (1-19, 20 or 21) Fragment 2 (20, 21, or 22-124) trypsin Ribonuclease ————> “‘I?Nase-T” 60°C Residues (1-31) attached to residues (34-124) by a disulfide bond Fic. 7. The limited cleavage of bovine pancreatic ribonuclease with sub- tilisin, elastase, and trypsin to yield active derivatives. The products produced by elastase and subtilisin may be separated into two chains which may be recom- bined through noncovalent interactions to yield full activity. In the trypsin product, the two stretches of sequence are held together through an SS bond and, after separation by reduction of this and the other 3 SS bonds, do not recombine cor- rectly upon SH oxidation. Both examples in the first group have been examined with respect to the stability of their conformations to conditions favoring disulfide interchange (Givol et al., 1965). Whereas the precursor zymogen chymotrypsinogen, a single-chained protein, is quite stable to sulfhy- dryl reagents and to the action of the disulfide rearranging enzyme mentioned earlier, its product of activation, chymotrypsin, is rapidly inactivated under such conditions, through “scrambling” of its di- sulfide bonds. One may conclude, therefore, that the information in the three polypeptide chains of the active protease is not sufficient to determine the correct structure and half-cystine pairing that one finds in this “derived” protein. A similar inactivation and structural disorganization occurs with insulin. This phenomenon led us (Givol e¢ al., 1965) to suggest that insulin, like chymotrypsin, might be synthesized as a “proinsulin” in 14 CHRISTIAN B, ANFINSEN IONE HIETCE NOSE IEE OIO MAR seek LEDUC LEN UEN AMEE ED ean acjenernieed ALA EDEN TILE EEIE IE IEEE ERO) OEE TE NUL ) 149 HOSTED OID YEE MENIAL IEN come Fic. 8. The amino acid sequence of an extracellular nuclease of Staphylococ- cus aureus, Specific pomts of cleavage, during digestion in the presence of deoxy- thymidine-3',5’-diphosphate and calcium ions by trypsin (T), chymotrypsin (C), and subtilisin (S$) are indicated by the arrows. “9 ! 1 5 ‘ 6 fo NH - [ALA] THR (cen (HR LVS\v5) Leu GLN)— COOH 6 ala 149 nH, -(LysYLEU mee GLY ) COOH oy ate nts (C3 Cedar L13- coon 1-H] omen a8 RI ORCS) Baa. coon P2-LYS HI Iv 149 ag my 30 me KD @)-com) | wD a) -com Pb Fic, 9. The formation of “nuclease-T” during the limited trypsin cleavage of staphylococcal nuclease (see also Fig. 8). As discussed in the text, fragments P2 and P3 associate, noncovalently, in solution to form an enzymatically active complex. FOLDING OF PROTEINS 15 which the normal chains are connected through a linking peptide joining the COOH-terminal residue of one with the NH,-terminus of the other. Following the properly directed pairing of half-cystine residues, the linking peptide might then be removed by a proteolytic process to yield the interchange-prone hormone. The recent discovery by Steiner and his colleagues of such a “proinsulin” molecule (Steiner, 1967), lends strong support to the general idea that thermodynamic instability of the structure of a protein indicates a precursor-product CONNECTING PEPTIDE Fic. 10. The structure of porcine proinsulin, including the amino acid se- quence of the connecting peptide that joins the B chain to the A chain. Courtesy of Drs. Chance, Ellis, and Bromer, Eli Lilly Co., Indianapolis, Indiana (Chance et al., 1968). relationship involving deletion of essential information. The structure of the porcine proinsulin is given in Fig. 10 (Chance et al., 1968). Ii is striking that recombination of the two reduced chains of insulin itself, through disulfide bond formation, can only be made to take place in high yield when certain ingenious chemical manipulations are employed in the process that favor the formation of the desired SS bonds. We have already discussed the case of RNase-S. This disulfide 16 CHRISTIAN B, ANFINSEN bonded protein could be studied by the estimation of the degree of “scrambling” of SS bonds under interchange conditions. A closely related phenomenon has recently been observed with another pro- tein which lacks SS bonds. Staphylococcal nuclease, whose structure (Taniuchi et al., 1967a; Cusumano e¢ al., 1968) is shown in F ig. 8, may be subjected to a limited proteolytic cleavage with trypsin, chymotrypsin, or subtilisin, when the digestion is carried out in the presence of calcium ions and a tightly bound substrate analog, deoxythymidine-3’,5’-diphosphate (Taniuchi et al., 1967b). These ligands stabilize the structure in a manner that restricts peptide bond cleavage to those specific bonds indicated in Fig. 9. The two large fragments resulting from trypsin attack may be separated from one another and, upon mixing in solution, regenerate the full activity of the original nuclease-T. The dissociation constant of the P2-P3 complex is approximately 10”, indicating a very precise and strong set of noncovalent interactions between the two peptide fragments (Taniuchi and Anfinsen, 1968). The y-globulins constitute a class of multichained proteins which are stable to disulfide interchange, in contrast to insulin and chymo- trypsin. We have already described the experiments of Haber (1964), Whitney and Tanford (1965), and Freedman and Sela (1966), which clearly show that a precise, antigen-specific structure is determined by the amino acid sequences of the two kinds of component chains. The stability to SS interchange, and the “informational sufficiency” may be explained by assuming that the sequences of the light and heavy chains are coded for by closely related genes and that the complete y-globulin molecule is a disulfide-linked oligomer rather than a combination of basically different individual chains. Light chains and heavy chains recombine to form active antibody, even after reduction and carboxymethylation of the half-cystine residues in- volved in interchain bonding (Edelman et al., 1963). The introduction of such disulfide bonds may have been an event in the natural selection of divalent, precipitating antibodies. Whereas “derived” multichained proteins such as chymotrypsin and insulin are thermodynamically unstable, proteins such as p- galactosidase (Zipser, 1963; Steers e¢ al., 1965; Shifrin and Steers, 1967) (containing four identical subunits) and aldolase (Penhoet et al, 1967) or hemoglobin (Kawahara et al., 1965) (with four homologous subunits) are conformationally stable and exhibit revers- FOLDING OF PROTEINS 17 ible denaturation. The latter proteins presumably represent examples of oligomers of closely related chains whose sequences are determined by duplicated homologous genes. Their oligomeric states appear to be involved with mechanisms of metabolic control (Monod e¢ al., 1965). SUMMARY Let me summarize the points I have made about the way in which conformational order is achieved at the point of transition from the linear information of the genotype to phenotypic function. First, the amino acid sequence coded for by a genetic cistron in turn codes for a specific three-dimensional structure. This conversion from linearity to spatial organization appears to be a spontaneous process. The native proteins that we find in cells are the polypeptide translations of genetic information, arranged in a form possessing maximum thermodynamic stability under physicological conditions. A particularly important factor in the determination of tertiary structures seems to be the internal and external positioning of hydrophobic and hydro- philic side chains, respectively. Second, the solution of a functional problem in terms of the three- dimensional arrangement of a polypeptide chain permits subsequent evolutionary changes in sequence only through mutations that are consistent with maintenance of the geometry of the prototypic pro- tein. Although insufficient data now exist, we may expect to find that a particular protein, or class of related proteins, isolated from a variety of species may have very similar three-dimensional structures. Finally, an examination of the extents to which various natural and “derived” multichained proteins undergo reversible denaturation suggests that interruption or deletion of information in the poly- peptide chain of single-chained proteins is generally not permissible, and that only those multichained proteins that are made up of identical or genetically related subunits may be reversible denatured. 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