Semisynthetic Cytochrome C (Peptide Synthesis/Reconstitution/Heme Protein) LEON E

Proc. Natl. Acad. Sd. USA Vol. 74, No. 10, pp. 4248-4250, October 1977 Biochemistry Semisynthetic cytochrome c (peptide synthesis/reconstitution/heme protein) LEON E. BARSTOW*, ROBERT S. YOUNG*, EMEL YAKALI*, JOHN J. SHARPt, JEAN C. O'BRIENt, PHILLIP W. BERMANt, AND HENRY A. HARBURYt * Department of Chemistry, University of Arizona, Tucson, Arizona 85721; and t Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 Communicated by Martin D. Kamen, July 22, 1977

ABSTRACT Horse heart cytochrome c can be split with constitution process can be utilized effectively to simplify the cyanogen bromide into a heme peptide (residues 1-65) and a latter approach. Quite clearly, semisynthesis of the desired nonheme peptide (residues 66-104). In a process involving (i) complex formation between the two fragments and (ii) resto- analogs, using the reconstitution process to couple a synthetic ration of the severed peptide linkage, a fully active cytochrome segment to a nonsynthetic one obtained through the cleavage c preparation can be re-formed. Use has been made of this procedure, should be a substantially less formidable undertaking process to couple the heme peptide to peptide 66-104 synthe- than synthesis of the'analogs in their entirety. sized bythe Merrifield solid-phase procedure. The semisynthetic -We report here the results of a first effort in this direction. product formed in this manner is indistinguishable from re- Segment 66-104 of the protein from horse heart has been syn- constituted cytochrome c prepared with nonsynthetic peptide thesized by the Merrifield solid-phase technique and has been 66-104. linked to heme peptide 1-65 prepared by the cyanogen bro- It has been shown in studies of cytochrome c from horse heart mide cleavage process. The product formed in this manner that the following cleavage and reconstitution process can be displays full electron-transfer activity and is indistinguishable effected (1-3): from reconstituted cytochrome c prepared with nonsynthetic peptide 66-104 (5). Heme Heme F I Met CNBr F I Hse> + 1 65 104 1 65 66 104 MATERIALS AND METHODS [I] Peptide 66-104. Na-Boc-L-glutamic acid--y-benzyl ester rHemel ', Heme resin ester (Boc is t-butoxycarbonyl) with a substitution of 0.2 FI Hse> + ~~~~~~~~~fastF I Hse>: mmol/g was prepared according to the method of Gisin (6). The 1 65 66 104 1 65 66 104 subsequent coupling steps were based on the sequence of op- [2] erations indicated in Table 1 (7-9). The synthesis was begun Hemel Heme] s with 10 g of the substituted resin ester and utilized Na-Boc- F Hse>: sow- I Hse [3] L-amino acids (3:1 molar ratio) throughout. The coupling of 1 65 66 104 1 65 104 asparagine was effected with the p-nitrophenyl ester (ONp) The reassembly proceeds in two readily separable stages. In the derivative. The following side-chain protection was used: first of these, a 1:1 complex is formed (Reaction 2), and in stage Tyr(Bzl), Thr(Bzl), Asp(,3-OBzl), Glu(y-OBzl), Arg(Tosyl), and two the amino group of residue 66 reacts with the homoserine Lys(Ne-Cbz). (Bzl is benzyl ether, OBzl is benzyl ester, Tosyl lactone residue in position 65 to reestablish a molecule with an is o-toluenesulfonyl, and Cbz is benzyloxycarbonyl.) Amino acid intact peptide chain. Since the presence of a homoserine rather analyses were performed after the addition of every third res- than a methionine side chain in position 65 is without effect on idue. the electron-transfer activity and other major characteristics Anhydrous HF-was used for cleavage of the peptide-resin of the product, the net result is a re-formation of the parent linkage (10, 11). The protected peptide-resin ester (0.5 g) and heme protein. 0.5 ml of anisole were mixed at 00 in a Teflon reaction vessel, Other cytochromes c with a methionine residue in position and approximately 20 ml of HF, distilled from cobalt trifluo- 65 can be split and reconstituted in an entirely comparable ride, was then passed into the vessel. After 0.5 hr, the peptide fashion, and it has been demonstrated that hybrid cytochromes was removed by extraction with 0.2 M acetic acid, and the can be formed by coupling the heme peptide of the protein of sample was filtered and lyophilized. Renewed treatment of this one species to the nonheme peptide of another (4). Such hybrid material with anisole and HF (00, 0.5 hr), followed by extraction formation provides a convenient means of generating experi- with acetic acid and lyophilization, yielded a product that was mentally useful variations in amino acid sequence, and addi- dissolved in 0.2 M acetic acid and subjected to gel filtration tional differences in structure can be realized through use of through Sephadex G-50. Two well-resolved bands were ob- the reconstitution process in concert with chemical and enzy- served. Fractions corresponding to the first of these (27% of the matic modification techniques. However, since many of the crude peptide) were pooled, lyophilized, and used directly in more pivotal questions concerning the cytochromes c relate to the reconstitution experiments. the structural and functional contributions of side-chain groups Semisynthetic Cytochrome c. The preparation of heme which can be varied only by the application of synthetic pro- peptide 1-65 from horse cytochrome c (Reaction 1) and the cedures, special interest attaches to the possibility that the re- coupling of the heme peptide to peptide 66-104 (Reactions 2 and 3) were done as described (1-3). Synthetic peptide 66-104 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Abbreviations: Boc, t-butoxycarbonyl; BA, benzyl ether; OBzl, benzyl "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate ester; Cbz, benzyloxycarbonyl; ONp, p-nitrophenyl ester; Tosyl, o- this fact. toluenesulfonyl. 4248 Downloaded by guest on September 30, 2021 Biochemistry: Barstow et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4249 Table 1. Coupling procedure Table 2. Amino acid composition of semisynthetic cytochrome c Step Reagent Mixing time No. of times Residues per molecule Amino acid of protein* 1 Dichloromethane 1 min 3 2 Trifluoroacetic acid (25%) Aspartic acid 8.3 (8) in dichloromethane 30 min 1 Threonine 9.7 (10) 3 Dichloromethane 1 min 3 Glutamic acid 11.7 (12) 4 Triethylamine (10%) in Glycine 11.9 (12) dichloromethane 1 min 2 Alanine 6.8 (6) 5 Dichloromethane 1 min 3 Valine 3.4 (3) 6 Boc amino acid in di- Methionine 0.8 (1) chloromethane, followed Isoleucine 5.9 (6) by dicyclohexyl- Leucine 5.5 (6) carbodiimide*t 5 hr 1 Tyrosine 3.8 (4) 7 Dichloromethane 1 min 3 Phenylalanine 3.7 (4) 8 Ethanol 1 min 2 Lysine 19.7 (19) 9 Dichloromethane 1 min 3 Histidine 2.5 (3) Arginine 1.6 (2) * Coupling of Boc- asparagine-ONp was conducted in dimethyl- formamide (72 hr). The resin-peptide was washed three times with * Literature values in parentheses. dimethylformamide before and after the coupling. t For the last 20 amino acids, the coupling step was carried out twice. exhibit slightly different binding affinities for an antibody The reaction times were 24 hr and 5 hr, respectively. Each coupling population specific for a site near residue 65, where the re- step was followed by steps 7-9. constituted preparation contains a homoserine rather than a was added to the reduced heme peptide in 0.1 M sodium methionine residue (17). This observation has led to a careful phosphate buffer, pH 7.0. The solution was kept in the reduced examination of the antibody-binding properties of semisyn- form at room temperature for 24 hr, and then was concentrated thetic cytochrome c. As shown in Fig. 2, the semisynthetic with the use of Aquacide (Calbiochem), dialyzed against 0.02 molecule yielded results identical to those obtained with non- M sodium phosphate buffer, pH 8.0, and subjected to isoelectric synthetic reconstituted cytochrome c (17). focusing (pH 8-10). Maximum separation was obtained in 15-17 hr. Fractions corresponding in position to nonsynthetic DISCUSSION reconstituted cytochrome c (6%) were pooled, dialyzed against Progress in studies of the structure-function relationships of the 0.02 M ammonium acetate buffer, pH 8.0, and Iyophilized. cytochromes c has been limited by a lack of adequate infor- Amino Acid Analysis. Resin-bound peptides were hydro- mation concerning the interactive effects operative between lyzed with hydrochloric acid/propionic acid (1:1) in evacuated the heme group and the amino acid residues of the surrounding ampules for 3 hr at 1300. Other samples were hydrolyzed with crevice region of the molecule. These effects lie at the heart of constant-boiling hydrochloric acid in evacuated ampules for 20-24 hr at 110°. Analyses were conducted with a Beckman model 120C amino acid analyzer. Activity. Determinations of electron-transfer activity were performed polarographically with beef heart mitochondria depleted of endogenous cytochrome c (12-14). Spectra. Absorption and circular dichroism spectra were C determined with a Cary model 17 spectrophotometer and a E Cary model 61 circular dichroism spectropolarimeter. 0 Antibody Binding. Radioimmunoassays were performed EI with antibodies from rabbits treated with horse cytochrome c (15-17). 25I-Labeled cytochrome c was prepared by the pro- cedure of Morrison et al. (18). .N C a0) RESULTS x 0 Amino acid analysis of the semisynthetic protein yielded the results indicated in Table 2. The absorption and circular dichroism spectra, as in the case of the nonsynthetic reconstitution product, corresponded closely to those of ferri- and ferrocytochrome c (2). Further evidence that the semisynthetic product closely re- sembles the parent heme protein is provided by the results of 0.2 0.4 0.6 0.8 1.0 electron-transfer activity determinations. As shown in Fig. 1, [Cytochrome i], ,uM the rates of oxygen consumption obtained in assays with mi- FIG. 1. Effects of cytochrome c (0), semisynthetic cytochrome tochondria depleted of endogenous cytochrome c were the c (0), and nonsynthetic reconstituted cytochrome c (A) on mito- same for semisynthetic cytochrome c as for the nonsynthetic chondrial succinate oxidation. Oxygen consumption was determined polarographically at 250 with the use of beef heart mitochondria reconstitution product and cytochrome c itself. subjected to depletion of endogenous cytochrome c. Each assay A fourth test that has been applied relates to the antibody- mixture contained, in a total volume of 1.6 ml: 0.01 M sodium phos- binding properties of the protein. It has been observed that phate buffer, pH 7.4; 0.25 M sucrose; 0.02 M sodium succinate; and cytochrome c and nonsynthetic reconstituted cytochrome c 1.1 mg of mitochondria. Downloaded by guest on September 30, 2021 4250 Biochemistry: Barstow et al. Proc. Natl. Acad. Sci. USA 74 (1977)

It V -I - - - I modated (4), but it is not known in detail to what extent alter- 100 ations of the naturally invariant residues can be tolerated. Presumably, the structural requirements with respect to these 80 positions are relatively stringent, and modifications that in-

C terfere significantly with establishment of the normal folding pattern will prevent productive complex formation and pep- 60 I 0 tide-bond restoration. This will limit the number of analogs obtainable by the reconstitution process, but the limitation is ._ 40k a helpful one. The most effective modifications for the study c of structure-function relationships are those that do not bring about extensive disruption of the normal packing arrangement 20 - of the molecule. To the extent that the establishment of a three-dimensional structure closely corresponding to that of the parent protein is also a requirement for peptide-bond refor- o 1 I I I I I I I mation, the reconstitution process fulfills a useful screening 0 4 8 12 16 20 role. [Competitor] / [label] FIG. 2. Radioimmunoassay of cytochrome c (0), semisynthetic cytochrome c (0), and nonsynthetic reconstituted cytochrome c (a). This investigation was supported by grants from the National Science Competition with 125I-labeled cytochrome c for binding to antibodies Foundation and the National Institutes of Health. elicited. against horse cytochrome c.

a number of the key characteristics of the c-type cytochromes 1. Corradin, G. & Hprbury, H. A. (1970) Biochim. Blophys. Acta to be in detail. Particularly useful, at this 221,489-496. but remain analyzed 2. Corradin, G. & Harbury, H. A. (1971) Proc. Nati. Acad. Sci. USA juncture, would be data delineating the role of (i) the aromatic 68,3036-3039. groups of the crevice, and (ii) the changes in ligand binding and 3. Corradin, G. & Harbury, H. A. (1974) Biochem. Biophys. Res. hydrophobic interaction known to occur upon oxidation-re- Commun. 61,1400-1406. duction of the prosthetic group (19, 20). 4. Corradin, G. & Harbury, H. A. (1974) Fed. Proc. 33, 1302. It is likely that the assembly of such data would be substan- 5. Barstow, L. E., Sharp, J. J., Young, R. S., O'Brien, J. C. & Harbury, tially facilitated by the examination of analogs containing la- H. A. (1977) Fed. Proc. 36,758. beled and/or structurally altered residues in the crevice region. 6. Gisin, B. F. (1973) Helv. Chim. Acta 56, 1476-1482. However, studies of this type can only be pursued once ade- 7. Merrifield, R. B. (1969) Adv. Enzymol. 32,221-296. selective preparative procedures have been devised. 8. Barstow, L. E., Cornelius, D. A., Hruby, V. J., Shimoda, T., Ru- quately pley, J. A., Sharp, J. J., Robinson, A. B. & Kamen, M. D. (1972) Approaches based on the reconstitution process, and especially in Chemistry and Biology ofPeptides, ed. Meienhofer, J. (Ann those designed along the lines of the present work, would appear Arbor Science Publishers, Ann Arbor, MI), pp. 231-233. to offer considerable promise in this regard. Through use of the 9. Sharp, J. J., Robinson, A. B. & Kamen, M. D. (1973) J. Am. Chem. semisynthesis procedure, any of the amino acid residues in Soc. 95,6097-6108. positions 66-104 of the protein can be isotopically labeled 10. Lenard, J. & Robinson, A. B. (1967) J. Am. Chem. Soc. 89, simply by incorporation of the label at the time of synthesis of 181-182. the nonheme peptide, and the same principle should apply to 11. Feinberg, R. S. & Merrifield, R. B. (1975) J. Am. Chem. Soc. 97, the introduction of various structurally modified residues. Since 3485-3496. the synthetic segment of the reconstituted molecule includes 12. Estabrook, R. (1967) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 10, pp. several of the aromatic groups of interest, and all of the residues 41-47. on the side of the heme where the strongly oxidation-state- 13. Blair, B. V. (1967) in Methods in Enzymology eds. Colowick, S. dependent coordination of methionine-80 occurs, the fact that P. & Kaplan, N. 0. (Academic Press, New York) Vol. 10, pp. the more immediate opportunities for analog formation relate 78-81. to this part of the chain rather than segment 1-65 represents, 14. Jacobs, E. & Sanadi, D. (1960) J. Biol. Chem. 235,531-534. if anything, an advantage. However, labels and structural al- 15. Farr, R. S. (1958) J. Infect. Dis. 103, 239-262. terations can probably be introduced to some extent-through 16. Patrick, J., Lindstrom, J., Culp, B. & McMillan, J. (1973) Proc. the use of standard chemical and enzymatic modification Nati. Acad. Sci. USA 70,3334-3338. procedures at the isolated heme peptide level-into region 1-65 17. Berman, P. W. (1977) Ph.D. Dissertation, Dartmouth College. 18. Morrison, M., Bayse, G. S. & Webster, R. G. (1971) Immuno- as well. of the protein chemistry 8, 289-297. The reconstitution process requires the formation of a 1:1 19. Harbury, H. A., Cronin, J. R., Fanger, M. W., Hettinger, T. P., complex in which the peptides are aligned in a manner suffi- Murphy, A. J., Myer, Y. P. & Vinogradov, S. N. (1965) Proc. Natl. ciently similar to that prevailing in the parent protein to permit Acad. Sci. USA 54,1658-1664. the reestablishment of a continuous peptide chain. Studies in 20. Harbury, H. A. & Marks, R. H. L. (1973) in Inorganic Bio- which hybrid cytochrome molecules were prepared show that chemistry, ed. Eichhorn, G. L. (Elsevier Publishing Co., Am- substantial variations in amino acid sequence can be accom- sterdam), pp. 902-954. Downloaded by guest on September 30, 2021