Protein semisynthesis

269

T I B S - December 1978

When semisynthetic RNase-S' is made, it is generally pure enough to allow characterization to a level expected for the fully native protein. For example, in the case of normal-sequence semisynIrwin M. Chaiken and Akira Komoriya thetic RNase-S', the product displays both full enzymic activity and normal 1:1 peptide-protein fragment association Semisynthesis provides a general approach to the engineering o f chemically altered proteins when derived by either classical [3] or and protein complexes. The use o f such analogues helps to understand how conformation solid-phase methods [5]. In the latter and function are specified by primary structure in proteins. case, structural identity with native complex has been substantiated by showing The demonstration that proteins can [3] and Scoffone's groups [4] have pro- that both semisynthetic and native RNasespontaneously regain a native state from vided methods for the chemical synthesis S' behave virtually the same in crystallone that is disordered [1] implies that the of fragment RNase-S-(I-20), which can ization and X-ray diffraction [5]. code of rules from which higher order be recombined non-covalently with native structure and function are derived can fragment RNase-S-(21-124) to yield Using semisynthetic sequence be deduced from information about semisynthetic RNase-S' (the 'prime' variants to learn about the primary structure. At present, there is denotes reconstituted fragment complex). native protein From the point of view of the protein an extensive knowledge of amino acid The semisynthetic complex can also be sequence and corresponding three- obtained from solid phase synthesized biochemist, the major benefit of being dimensional structure for many proteins: RNase-S-(l-20), usually requiring func- able to produce high quality semisynHence, there is the opportunity to correlate tional purification of the original crude thetic RNase-S' is that it provides the the chemical detail of amino acid sequen- synthetic peptide by complementation basis for the engineering of sequence ces with conformational and functional with RNase-S-(21-124) itself and sub- variants. Indeed, the list of semisynthetic features of high-resolution protein models, sequent isolation of the reconstituted replacement and deletion analogues of with the goal of generating ideas as to species [5]. Each synthetic approach has RNase-S' produced so far is quite extenhow these features arise. Such ideas can both advantages and shortcomings for sive [2,6-8] and continues to grow. be tested by studying the effects of per- RNase-S', but the end result in both As indicated by the view obtained by turbation of particular sequences that cases is dependable production of a X-ray diffraction analysis (Fig. 2), the semisynthetic non-covalent complex RNase-S-(l-20) fragment when in the we select in the molecular models. One technique which offers unique and, where desirable, the isolated syn- intact non-covalent complex is extensively =-helical (residues 3 through 13) with advantages in 'protein mutation' is thetic RNase-S-(1-20) species itself. chemical synthesis. By this method, proteins altered at chosen loci can be prepared for detailed characterization. Although the state of the art still limits the size of polypeptides which can be synthesized totally, the related approach of semisynthesis provides a means of preparing relatively small peptide fragments, incorporating these into intact protein species, and thereby studying effects of synthetic modification on macromolecular properties (Fig. 1). The results so far demonstrate the potential use of semisynthesis not only to study properties of the individual proteins but also to test general mechanistic models for how proteins operate in folding, conformationai stabilization, and biological function.

Protein semisynthesis

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Native rote (

Semlsynthetie ribonudease-S' The case of bovine pancreatic ribonuclease-S [2] offers a well-developed example of the semisynthesis approach. The now-classical efforts of Hofmann's The authors are at the Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, MD. 20014. U.S.A.

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Fig. 1. Scheme showing the various aspects of protein semisynthesis.

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T I B S - December 1978

270 several key side chains either in the active site (as His-12) or making stabilizing contacts with RNase-S-(21-124) (as Pbe-8 and Met-13). All of these categories, of residues have been the object of investigation by synthesis. In retrospect, one of the abiding interests in analogue work so far has been to study those residues which are important for the direct enzymic function of the native protein. For example, the mode of direct function of His-12 in RNase catalysts, first implied by alkylation studies of RNase-A [9], has been refined extensively by the preparation of semisyntbetic analogues (for summaries, see [2,8,10]). The message from all such studies is that when the chemical nature of the imidazolyl group is altered, especially by changing pK, the result is suppression of catalytic activity. To be sure, occasional independent evidence [11] has suggested that significant RNaselike activity can be displayed by derivatives devoid of His-12. However, only trace amounts of activity can be observed with [4-fluoro-His 1=, des 16-20] semisynthetic RNase-S' (where the imidazolyl PKa at position 12 is about 2, versus 6 for the normal histidine) against both step I and step 2 substrates [12, 13]. These data, combined with the findings for this analogue of a highly normal conformation, ribonuelease-like recognition of substrates and inhibitors [14], and a normal pKa for His-ll9 (6.4 [15] versus 6.30 for native (with corrected assignment)), emphasize the crucial participation of His-12 in the major catalytic processes of native RNase. Results such as those for the 4-fluoro-His analogue support models for the catalytic mechanism of pancreatic RNase in which His-12 has a requisite acid-base function [2]. The information content for RNase-S(1-20) necessary for the conformational integrity of RNase-S also has been the subject of investigation. The results so far indicate that the NH=-terminal or-helix normally contributed by residues 3 to 13 seems intimately involved in providing the proper conformation for a productive peptide-protein interaction. Also involved in this interaction are such specific side chains as the aromatic residue at position 8 (normally a Pbe which is replaceable by Tyr but not by Ile or Ala [6]). In contrast, the non-essentiality of residues 16 through 20 for conformation is evident, based on data for [des 16-20] semisynthetic RNase-S', which show not only full activity .with a 1:1 peptideprotein complex but also native-like

behavior in crystallization and X-ray diffraction [7,16]. Future efforts in designing semisyn"thetic analogues for characterizing RNase-S should benefit from two basic factors. (a) Current information should allow reasonable predictions to be made as to what effects particular synthetic manipulations might have on the properties of the RNase-S complex. Such predictions can be based on the extensive data already compiled from studies not only of the native bovine protein and previously studied semisynthetic bovine sequence variants but also of natural sequencevariant RNases of different but closely related species [17]. These data can be considered along with the increased understanding of tendencies of amino acids to conform to particular secondary structures [18]. (b) As expected for native RNase-S, it should be possible to study a wide range of characteristics for semisynthetic species. This expectation has been extended to high resolution structural analysis by X-ray diffraction of large crystals, which can be grown from solid phase-derived [5,16] and classically-derived [19] products. Given these factors, the cyclical process indicated in Fig. I can be a useful way to decipher particularly complex relationships between RNase sequence and properties of the folded protein.

that a functionally and conformationally viable 'model' RNase-S analogue can be made in which the peptide component is poly-alanine (predicted to supply the high helix propensity presumed necessary) with a few side chains (such as Pbe-8 and His-12) predicted to be necessary to contribute a sufficient number of critical contacts and functional elements. We expect that further efforts of this type of study will lead to a more wideranging experimental documentation of the interplay between the chemical detail of an amino acid sequence and its conformational propensity in generating a functional protein.

The incorporation of non-perturbing probes Semisynthesis also can allow the incorporation of non-perturbing probes into particular loci of proteins. For example, NMR studies of zaC-enriched RNase analogues have shown that conformational transitions at particular residues can be monitored by following the chemical shifts of individual carbon atoms [22]. This possibility at present is being applied in the authors' laboratory (with [xsC-Phe 8]-semisyntbetic RNase-S')

Studies of general properties of proteins Beyond its use to describe the RNase molecule itself, the semisyntbetic RNase-S' system can serve as a model for studying codification of function and conformation in primary structure for all proteins. For example, this system has been used to obtain experimental verification for empirically-derived rules describing the propensity of certain amino acid sequences for the ~-hellcal conformation [20]. For a series of analogues of the semisynthetic complex in which the normally helix-involved Glu-9 was replaced by either Leu or Gly, the non-covalent association constants of the respective complexes were generally proportional to the helix propensity parameters predicted for the residues at position 9 [21]. The data emphasize the general relationship between helix propensity and the conformational stability of the RNase-S complex. In an extension of this study in the authors' laboratory, it has been found

\ HIS 12 PHE 8

LYS 1

Fig. 2. Representation of the 1-15 sequence of ribonuclease, in the conformation this segment assumes in native RNase-S [2]. The tail of residues 16-20, not shown, is largely disordered in this

complex

T I B S - D e c e m b e r 1978

to study the relationship between temperature-dependent overall unfolding and temperature-dependent unfolding at a particular residue in a helical segment. Other applications of non-covalent semisynthesis

Recent studies indicate that the semisynthesis approach should have general application to many protein species. Semisynthesis involving incorporation o f synthetic peptide into a protein entity by only non-covalent interactions (as for RNase-S) has been applied in several cases for which viable complexes can be produced by limited proteolysis of native protein• For staphylococcal nuclease, both a trypsin-derived complex, nuclease-T (containing fragments of residues 6-48 and 49 or 50-149) and a second complex consisting o f trypsin fragment (1-126) and cyanogen bromide fragment (99-149) have been subjects of such work [23,24]. In the case of nuclease-T, semisynthetic complex of high enzymic activity has been isolated containing the synthetic peptide (6-47) [25]. Further, conformational and active site features have been investigated by synthetic replacement and deletion [23,26,27]. The case of synthetic-(6-47) peptide, that corresponds to a sequence forming a large region of antiparallel }8-pleated sheet in the intact protein, has allowed (analogous to the RNase-S ,,-helix studies alluded to above) some initial correlations to be made between sequence and E-pleated sheet stabilization [27]. Extensive semisynthesis studies also have been earried out for a 'C-terminar complex derived from pepsin-inactivated panc'~eatic ribonuclease [28]. Development and application of covalent semisynthesis

In view of the limited occurrence o f viable non-covalent protein fragment complexes, efforts have been made to develop general methods for covalent introduction of synthetic peptide fragments into proteins. Pioneering efforts in designing approaches to the selective protection of native protein for fragment production and restitching o1" protected synthetic and native peptides have been made with such proteins as insulin, cytochrome c, phospholipase As, and myogiobin (for specific discussions, see other papers in [8]). In a somewhat specialized case, sterically facilitated restitching has been used successfully to prepare active, covalently intact [Hser-65] cytochrome c from the initial non-

271 covalent complex of cyanogen bromide fragment (1-65, heine) and synthetic(66--104) [29]. A similar semisynthesis has been achieved for pancreatic trypsin inhibitor [30]. Whereas both of these results depend on the presence of an activated homoserine lactone, the use • of enzymes to promote sterically enhanced semisynthesis has been effected for soybean trypsin inhibitor [31] and recently applied to the restitching of RNase-S to RNase-A [32]. This latter development could be a valuable adjunct in future studies using RNase-S semisynthesis. Future prospects

6 Scoffone, E., Marchiori, F., Moroder, L., Rocchi, R. and Borin, G. (1973) in Medicinal Chemistry II1, Milan, Special Contributions (Pratesi, P., ed.), pp. 83-104,

Bunerworth and Co., London 7 Finn, F. M. and Hofmann, K. (1973) Accts. Chem. Res. 6, 169-176 8 Chaiken, I. M. (1978) in Semisynthetic Peptides and Proteins (Offord, R. E., and DiBello, C., eds), pp. 349-364, Academic Press, London 9 Crestfield, A. M., Stein, W. H. and Moore, S. (1963) J. Biol. Chem. 238, 2413-2420 10 van Batenburg, O. D., VoskuyI-Holtkamp, I., Schattenkerk, C., Hoes, K., Kerling, K. E. and Havinga, E. (1977) Biochem. J. 163, 385-387 II Gutte, B. (1975) J. Biol. Chem. 250, 889-904 12 Dunn, B. M., DiBello, C., Kirk, K. ~L., Cohen, L. A. and Chaiken, I. M. 0974) J. Biol. Chem. 249, 6295-6301 13 Komoriya, A., A m m o n , H. L. and Chaiken,

The semisynthetic work to date on RNase-S indicates the possibility of gaining important insights not only into I. M., in preparation the function of specific parts of an amino 14 Taylor, H. C. and Chaiken, I. M. (1977) acid sequence in a particular protein J. Biol. Chem. 252, 6991-6994 but also into general theoretical ques15 Dunn, B. M. and Chaiken, [. M. (1974) in tions regarding the relationship between Peptides 1974 (Wolman, Y., ed.), pp. 299sequence and the higher order properties 309, John Wiley and Sons, Inc., New York of folded conformation and biological 16 Chaiken, L M., Taylor, H. C. and Ammon, activity. Given the increase of achieveH. L. (1977) J. Biol. Chem. 252, 5599-5601 ments in semisynthesis, it should be 17 Lenstra, J. A., Hofsteenge, J. and Beintema, J. J. (1977) J. Mol. Biol. 109, 185-193 possible to study the formation of a variety of structural entities, such as 18 Anfinsen, C. B. and Scheraga, H. A. (1975) in Adv. Prot. Chem. (Anfinsen, C. B., Edsall, ,`-helices (as in the case of RNase-S), J. T. and Richards, F. M., eds), Vol. 29, /3-pleated sheets (as for nuclease-T), pp. 205-300, Academic Press, New York /3-bends, and contact surfaces, at the level of the intact protein• The organ- 19 Valle, G., Zanotti, G., Filippi, B. and Del Pra, A. (1977) Biopolymers 16, 1371-1376 ization and operation of various active 20 Dunn, B. M. and Chaiken, I. M. (1975) site types also should be subject to deJ. MoL Biol. 95, 497-511 tailed structure-function characterization, 21 Chou, P. Y. and Fasman, G. D. (1974) especially by the minimal modification Biochemistry 13, 211-222 possible through semisynthesis. The 22 Chaiken, I. M., Freedman, M~ H., Lyerla, broader understanding of native proteins J. R., Jr. and Cohen, J. S. (1973) J. Biol. Chem. 248, 884--891 and the rules by which they function ultimately could allow for systematic 23 Ontjes, D. A. and Anfinsen, C. B. (1969) Proc. Nat. Acad. Sci. U.S.A. 64, 428-435 engineering attempts to modify protein specificity and to produce simplified "24 Parikh, I., Corley, L. and Anfinsen, C. B. (1971) J. Biol. Chem. 246. 7392-7397 model systems possessing biological 25 Chaiken, I. M. (1971) J. Biol. Chem. 246, activity. 2948-2952 26 Chaiken, I. M. and Anfinsen, C. B. (1971) References J. Biol. Chem. 246, 2285-2290 1 Epstein, C. J., Goldberger, R. F. and Anfin- 27 Chaiken, I. M. (1972) J. Biol. Chem. 247, sen, C. B. (1963) Cold Spring Harbor Symp. 1999-2007 Quant. Biol. 27, 439--449 28 Lin, M. C., Gutte, B., Moore, S. and Merri2 Richards, F. M. and Wyckoff, H. W. (1971) feld, R. B. (1970) J. Biol. Chem. 245, 5169in The Enzymes (Boyer, P., ed.), Vol. IV, 5170 Third Edn, pp. 647-806, Academic Press, 29 Barstow, L. E., Young, R. S., Yakali, New York E., Sharp. J. J., O'Brien, J. C., Berman, 3 Hofmann, K., Visser, J. P. and Finn, F. M. P. W. and Harbury, H. A. (1977) Proc. (1969) J. Am. Chem. Soc. 91, 4883-4887 Nat. Acad. Sci. U.S.A. 74, 4248--4250 4 Scoffone, E., Marchiori, F., Marzotto, A. and Rocchi, R. (1967) in Peptides 1967 30 Dyekes, D. F., Creighton, T. E. and Sheppard R. C. (1978) Int. J. Peptide Protein Res. (Beyerman, H. C., Van de Linde, W. and 1 i, 258-268 Masen van den Brink, W., eds), pp. 280-285, 31 Kowalski, D. and Laskowski, M., Jr. North Holland Pub. Co., Amsterdam (1976) Biochemistry 15, 1309-1315 5 Pandin, M., Padlan, E. A., DiBello, C. and Chaiken, I'. M. (1976) Proc. Nat. Acad. 32 Homandberg, G. A. and Laskowski, M., Sci. U.S.A. 73, 1844-1847 Jr. (1978) Fed. Proc. 37, 1813