Selenocysteine in native chemical ligation and expressed protein ligation.

L-Selenocysteine (Sec or U) has been called the “21st amino acid”.1 Like the twenty common amino acids, selenocysteine is inserted during the translation of mRNA and has its own tRNASec and codon, UGA. This codon also serves as the opal stop codon. Decoding a UGA codon as one for selenocysteine requires a special structure in the 3′ untranslated region of the mRNA called a selenocysteine insertion sequence (SECIS) element. Because eukaryotic and prokaryotic cells use a different SECIS element to decode UGA as selenocysteine, the production of eukaryotic selenocysteine-containing proteins in prokaryotes is problematic.2 Here, we describe a general semisynthetic route to proteins containing selenocysteine.3,4 In “native chemical ligation”, the thiolate of an N-terminal cysteine residue in one peptide attacks a C-terminal thioester in another peptide to produce, ultimately, an amide bond between the two peptides (Scheme 1).5 “Expressed protein ligation” is an extension in which the C-terminal thioester is produced by using recombinant DNA (rDNA) technology.6 We reasoned that selenocysteine, like cysteine, could effect both native chemical ligation and expressed protein ligation, and thereby provide a means to incorporate selenocysteine into proteins. We used AcGlySCH2C(O)NHCH3 as a model thioester to test the feasibility of using selenocysteine in native chemical ligation.7 Reaction with cystine ((CysOH)2) in the presence of the reducing agent tris-(2-carboxyethyl)phosphine (TCEP) produced AcGlyCysOH, as well as some (AcGlyCysOH)2. When selenocystine ((SecOH)2) was used in the same reaction, the product was (AcGlySecOH)2. A selenolate (RSe-) is more nucleophilic than is its analogous thiolate (RS-).9 Moreover, the pKa of a selenol (RSeH) is lower than that of its analogous thiol (RSH).9a,10 These properties suggested to us that native chemical ligation with selenocysteine could be more rapid than with cysteine, especially at low pH. To test this hypothesis, we used the chromogenic thioester AcGlySC6H4-p-NO2 (1; Scheme 2) to determine the rate of native chemical ligation as a function of pH.11 The resulting pH-rate profile is shown in Figure 1. Reaction with selenocysteine is 103fold faster than with cysteine at pH 5.0. Thus, native chemical ligation with selenocysteine can be chemoselective.12 Having demonstrated the effectiveness of selenocysteine in native chemical ligation, we next set out to explore its utility in expressed protein ligation. As a model protein, we chose ribonuclease A (RNase A; EC 3.1.27.5; Figure 2), which has been the object of much seminal work in protein chemistry.14 RNase A has 8 cysteine residues that form 4 disulfide bonds in the native * To whom correspondence should be addressed. Telephone: (608) 2628588. Fax: (608) 262-3453. E-mail: [email protected]. † Department of Biochemistry. ‡ Department of Chemistry. (1) (a) Bock, A.; Forchhammer, J.; Heider, W.; Leinfelder, G.; Veprek, B.; Zinoni, F. Mol. Microbiol. 1991, 5, 515-520. For other reviews, see: (b) Odom, J. D. Structure Bonding 1983, 54, 1-26. (c) Low, S. C.; Berry, M. J. Trends Biochem. Sci. 1996, 21, 203-208. (d) Stadtman, T. C. Annu. ReV. Biochem. 1996, 65, 83-100. (2) Arner, E. S. Sarioglu, H.; Lottspeich, F.; Holmgren, A.; Bock, A. J. Mol. Biol. 1999, 292, 1003-1016. (3) For other means to incorporate selenocysteine into semisynthetic and synthetic proteins, see: (a) Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1989, 111, 4513-4514. (b) Fiori, S.; Pegoraro, S.; Rudolph-Bohner, S.; Cramer, J.; Moroder, L. Biopolymers 2000, 53, 550-564. (4) For a means to produce selenopeptide libraries on the surface of M13 bacteriophage, see: Sandman, K. E.; Benner, J. S.; Noren, C. J. J. Am. Chem. Soc. 2000, 122, 960-961. (5) (a) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs Ann. Chem. 1953, 583, 129-149. (b) Dawson, P. E.; Muir, T. W.; ClarkLewis, I.; Kent, S. B. Science 1994, 266, 776-779. For a recent review, see: (c) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923-960. (6) (a) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705-6710. For reviews, see: (b) Cotton, G. J.; Muir, T. W. Chem. Biol. 1999, 6, R247-R256. (c) Evans, T. C., Jr.; Xu, M.-Q. Biopolymers 2000, 51, 333-342. (7) AcGlySCH2C(O)NHCH3 was synthesized as described by Nilsson B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939-1941. We find N-methylmercaptoacetamide to be a superb thiol for effecting both native chemical ligation and expressed protein ligation. (8) L-Selenocystine was synthesized from elemental selenium and â-chloroL-alanine as described by Tanaka, H.; Soda, K. Methods Enzymol. 1987, 143, 240-243. Ligation reactions were performed at (23 ( 2) °C under Ar(g) in 0.10 M Tris-HCl buffer (pH 8.0) containing AcGlySCH2C(O)NHCH3 (25 mM), TCEP (25 mM), and cystine or selenocystine (12.5 mM). (9) (a) Huber, R. E.; Criddle, R. S. Arch. Biochem. Biophys. 1967, 122, 164-173. (b) Pearson, R. G.; Sobel, H.; Songstad, J. J. Am. Chem. Soc. 1968, 90, 319-326. (c) Pleasants, J. C.; Guo, W.; Rabenstein, D. L. J. Am. Chem. Soc. 1989, 111, 6553-6558. (d) Singh, R.; Whitesides, G. M. J. Org. Chem. 1991, 56, 6931-6933. (e) Gorlatov, S. N.; Stadtman, T. C. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8520-8525. (f) Lee, S.-R.; Bary-Noy, S.; Kwon, J.; Levine, R. L.; Stadtman, T. C.; Rhee, S. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2521-2526. (g) Zhong, L.; Holmgren, A. J. Biol. Chem. 2000, 275, 18121-18128. (10) Arnold, A. P.; Tan, K. S.; Rabenstein, D. L. Inorg. Chem. 1986, 25, 2433-2437. (11) Ligation assays were performed at (23 ( 2) °C under Ar(g) in 0.10 M buffer containing thioester 1 (10 μM), TCEP (0.10 mM), and cystine (20 μM-0.50 mM) or selenocystine (1.0-20 μM). Rate ) ∂[HSC6H4-p-NO2]/∂t ) kobs[CysOH or SecOH] kH2O was determined by using ) 11, 230 M-1 cm-1 for p-nitrothiophenolate at 410 nm. (12) The template-directed ligation of a phosphoroselenol and iodoribose is approximately 4-fold faster than that of an analogous phosphorothiol at pH 7.0 (Xu, Y.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 9040-9041). We observe a similar difference in Se vs S reactivity at high pH (Figure 1). (13) Danehy, J. P.; Parameswaran, K. N. J. Chem. Eng. Data 1968, 13, 386-389. (14) Raines, R. T. Chem. ReV. 1998, 98, 1045-1065. Scheme 1