Báo cáo khoa học: Inhibitory activity of double-sequence analogues of trypsin inhibitor SFTI-1 from sunflower seeds: an example of peptide splicing docx

Inhibitory activity of double-sequence analogues of trypsin inhibitor SFTI-1 from sunflower seeds: an example of peptide splicing Anna Łe˛ gowska 1 , Adam Lesner 1 ,El _ zbieta Bulak 1 , Anna Jas ´ kiewicz 1 , Adam Sieradzan 1 , Marzena Cydzik 2 , Piotr Stefanowicz 2 , Zbigniew Szewczuk 2 and Krzysztof Rolka 1 1 Faculty of Chemistry, University of Gdan ´ sk, Poland 2 Faculty of Chemistry, University of Wrocław, Poland Introduction Trypsin inhibitor SFTI-1, the smallest member of the family of Bowman–Birk inhibitors (BBIs), has been found in sunflower seeds [1]. This homodetic peptide consists of 14 amino acid residues and its structure is stabilized by a disulfide bridge (Fig. 1). The reactive site P 1 -P 1 ¢ of this peptide is located between Lys5-Ser6. As a result of the high sequential and structural homology of SFTI-1 with the binding loop of the canonical inhibitors of the BBI family, SFTI-1 forms a complex with the target enzyme in stoichiometric ratio of 1 : 1. As reported by Marx et al. [2], upon the incu- bation with trypsin, the ratio of native SFTI-1 to its acyclic permutant with hydrolyzed P 1 -P 1 ¢ is approxi- mately 9 : 1. As a result of its small size, very strong trypsin inhibitory activity and circular backbone scaf- fold (i.e. well defined 3D structure, displayed b-hairpin motive), SFTI-1 has attracted interest ever since its dis- covery. Recent studies on SFTI-1 are summarized in three reviews [3–5]. Because small proteinaceous inhibi- tors are of commercial interest, SFTI-1 soon became an attractive template for the design of new protease inhibitors with potential use as therapeutic and agro- chemical agents. Subsequent to the discovery of SFTI-1, several stud- ies have shown that the presence of both cycles in the inhibitor molecule is not essential for its activity. A monocyclic analogue of SFTI-1, containing only the disulfide bridge, appeared to have trypsin inhibitory activity matching that of the wild-type SFTI-1 [6–8]. In addition, it displayed proteinase resistance similar to that of the parent compound [9]. Another analogue, containing only head-to-tail cyclization, ([Abu 3,11 ] Keywords inhibitors; mass spectrometry; peptide splicing; serine proteinases; SFTI-1 Correspondence A. Łe˛ gowska, Faculty of Chemistry, University of Gdan ´ sk, Sobieskiego 18, 80-952 Gdan ´ sk, Poland Fax: +48 5852 3472 Tel: +48 5852 3359 E-mail: legowska@chem.univ.gda.pl (Received 4 February 2010, revised 10 March 2010, accepted 15 March 2010) doi:10.1111/j.1742-4658.2010.07650.x Four 28-amino acid peptides were synthesized whose sequences comprised two molecules of trypsin inhibitor sunflower trypsin inhibitor 1 (SFTI-1) bound through a peptide bond. The peptides in their reactive positions (5 and 19 of the peptide chain) contain two Lys ([KK]BiSFTI-1) and two Phe ([FF]BiSFTI-1) residues, along with a combination of the amino acid residues named thereafter [KF]BiSFTI-1 and [FK]BiSFTI-1. Association constants of the analogues determined with trypsin and chymotrypsin, respectively, indicated that they were potent inhibitors of cognate protein- ases. An MS study of the associates revealed that incubation of the com- pounds with the proteinases resulted in cutting out a fragment of the peptide chain to restore the native monocyclic molecule of SFTI-1 or its analogue [Phe 5 ]SFTI-1. This process, analogous to that of the DNA and protein splic- ing, can be referred to as ‘peptide splicing’. Abbreviations BBI, Bowman–Birk inhibitor; ESI, electrospray ionization. FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS 2351 SFTI-1) displayed trypsin inhibitory activity that was only 2.5-fold lower than the wild-type inhibitor [6]. As reported by Korsinczky et al. [8,9], solution structures of such monocyclic SFTI-1 analogues are remarkably similar to the solution and the crystal structures of the wild-type SFTI-1. A higher structural flexibility of [Abu 3,11 ]SFTI-1 compared to that of SFTI-1 is com- patible with its lower activity and higher hydrolysis rate. In the wild-type of SFTI-1, a substrate specificity P 1 position [1] is occupied by Lys residues. For this reason, SFTI-1 and its monocyclic analogues with Lys5 were demonstrated to be strong trypsin inhibitors [6], whereas their chymotrypsin inhibitory activity was three orders of magnitude lower when association con- stants (K a ) with appropriate serine proteinases were used as a measure of their strength [7]. Monosubstitu- tion of Lys5 by Phe reversed the SFTI-1 specificity. Thus, [Phe 5 ]SFTI-1 did not inhibit trypsin but exhib- ited strong chymotrypsin inhibitory activity with K a = 2.0 · 10 9 m )1 [10]. When designing compounds of commercial impor- tance, the aim is to reduce their size and simplify the original structure of a naturally occurring compound (e.g. protein). Bearing in mind the potential applica- tions of BBIs, and considering the results presented above, we designed four SFTI-1 analogues based on the double-sequence of the wild-type inhibitor. The primary structures of dimeric SFTI-1 analogues are shown in Fig. 1. In all of the compounds, two sequences are bound by a peptide bond formed between the C-terminal a-carboxyl group of Asp in the first molecule and the N-terminal a-amino group of Gly in the second one. To form one disulfide bond only, two Cys residues located in the middle of the peptide chain (positions 11 and 17) were replaced by their structural counterparts of a-aminobutyric acid (Abu) residues, whereas the remaining two formed a disulfide bond. The dimeric SFTI-1 analogues differ at positions 5 and 19. Our synthesized analogues, [KK]BiSFTI-1 (5) and [FF]BiSFTI-1 (6), as well as [KF]BiSFTI-1 (7) and [FK]BiSFTI-1 (8), contain Lys and Phe in positions 5 and 19, in addition to combina- tions of both amino acid residues. Our intention was to design low-molecular compounds containing two reactive sites, with the second one located between positions 19 and 20. We assumed that these analogues would be able to form complexes with trypsin or chy- motrypsin with a stoichiometry of 2 : 1 (analogues of 6 and 7), whereas the two remaining analogues would inhibit both trypsin and chymotrypsin simultaneously and independently. Jaulent and Leatherbarrow [11] reported the synthesis and kinetic studies of a bicyclic and bifunctional proteinase peptidic inhibitor consist- ing of 16 amino acids. The inhibitor was designed by combining two binding loops of BBI. As postulated by Jaulent and Leatherbarrow [11], the size of the inhibi- tor was incompatible with the simultaneous binding of trypsin and chymotrypsin. We predicted that the size of 28 amino acid residues peptides might be sufficient to accommodate both enzyme molecules. Results and Discussion As indicated in Table 1, all four dimeric SFTI-1 per- mutants, with the exception of 8, incubated with tryp- sin, are potent inhibitors. The K a values for the compounds are approximately one order of magnitude lower than those for their monomeric counterparts. Surprisingly, [FK]BiSFTI-1 (8) did not block trypsin activity. This enzyme regained its activity (within A B Fig. 1. Chemical structures of (A) SFTI-1 and (B) synthesized analogues [KK]BiSFTI-1 (5), [FF]BiSFTI-1 (6), [KF]BiSFTI-1 (7) and [FK]BiSFTI-1 (8). An example of peptide splicing A. Łe˛ gowska et al. 2352 FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS 5 min) after incubation with 8, thus suggesting that 8 behaved as a substrate rather than an inhibitor. At the same time, this shows that the peptide bond between Lys19 and Ser20 (the reactive site) and the Arg 16 - Abu 17 bond are both rapidly hydrolyzed by the enzyme (Fig. 2) and the hydrolysis products are quickly released from the enzyme’s substrate pocket. It is interesting to note that, when compounds 7 and 8 were preincubated with one enzyme each and then their inhibitory activities were checked against another enzyme, they displayed inhibitory activity that was at least one order of magnitude higher. On the basis of these results, it can be assumed that compounds 7 and 8 inhibit two proteinases independently and simultaneously. We also found that each of the used chromogenic substrates was specific for one of the experimental proteinases and remained intact in the presence of the other enzyme. Consequently, the hypothesis that the inhibitory activity of the permu- tants in the presence of both enzymes could have been caused by experimental conditions can be ruled out. Indeed, we conducted the experiments under the condi- tions recently described by Jaulent and Leatherbarrow [11], who reported synthesis and kinetic studies on a bicyclic and bifunctional proteinase peptidic inhibitor consisting of 16 amino acids. The inhibitor (BiKF) was designed by combining two building loops of BBI and was able to inhibit both trypsin and chymo- trypsin independently but not simultaneously. This means that, after preincubation with one enzyme, it completely lost its inhibitory activity against the other one. As claimed by Jaulent and Leatherbarrow [11], the size of the inhibitor precluded the simulta- neous binding of both trypsin and chymotrypsin. The 28-amino acid peptides described in the present study are remarkably bigger, although they still remain rela- tively small compared to the proteinases, trypsin (23 284 Da) and chymotrypsin (25 225 Da). In this sit- uation, the hypothesis that dimeric SFTI-1 inhibitors could form either 2 : 1 or 1 : 1 : 1 complexes still needs further exploration. Unfortunately, based on the kinetic studies performed, it is impossible to determine the stoichiometry of the complexes formed by protein- ase(s) with dimeric SFTI-1 analogues. In an attempt to do so, we employed gel electrophoresis and HPLC with a size-exclusion column. Unfortunately, the results obtained in these experiments were not convinc- ing and are not discussed here. One of the methods of choice for studying noncova- lent complexes formed by proteins is MS with electro- spray ionization (ESI). An in-depth analysis of complexes formed between bovine pancreatic trypsin inhibitor and target proteinases was provided by Nesatyy [12], who also emphasized that a correlation between the solution and gas phase binding of the complexes was not straightforward. There was a not- oceable difference in the strength of the complexes formed in the aqueous and gas phase, whereas their stoichiometry was preserved. Figures 3 and 4 represent MS spectra of trypsin and chymotrypsin along with those recorded after their incubation with [KK]BiSFTI-1 (5) and [FF]BiSFTI-1 (6), respectively. The ESI spectra of bovine b-trypsin (Fig. 3A) exhibited two charge states of +9 and +10. The molecular mass of the enzyme calculated from the first peak was 23 322 Da, whereas the other peak cor- responded to a trypsin molecule with a trapped cal- cium ion. After incubation of 5 with trypsin, among the four peaks seen in the MS spectrum, those with m ⁄ z 2333.2753 and 2592.4874 were assigned to free trypsin, whereas the remaining two with m ⁄ z 2486.4686 and 2762.4797 revealed the appearance of a 1 : 1 complex of trypsin with monocyclic SFTI-1 (Fig. 3B). Essentially, an identical peak pattern was seen with an increasing incubation time of up to 20 h (data not shown). The MS spectrum of bovine a-chy- motrypsin (Fig. 4A) produced charge states of +10 and +11. The monoisotopic molecular mass (calculated using the SNAP procedure in the Bruker Data Analysis program; Bruker Daltonics, Bremen, Germany) of the proteinase derived from those peaks was 25 225 Da. In the spectrum of a 1 : 1 mixture of chymotrypsin and [FF]BiSFTI-1 (6) incubated for 30 min (Fig. 4B), two peaks were seen with charge states of +10 and +11, both representing a 1 : 1 complex between Table 1. Association constants (K a ) of SFTI-1 analogues based on the double-sequence of SFTI-1. Ch and T in parenthesis indicate that the inhibitory activity was determined after preincubation of the inhibitor with bovine a-chymotrypsin or bovine b-trypsin, respectively. ND, not determined (i.e. peptide unstable under the conditions used for K a determination). Number Analogue K a [M )1 ] Trypsin Chymotrypsin 1 SFTI-1 (wild) [6,7] 1.1 · 10 10 5.2 · 10 6 2 SFTI-1 a [6,7] 9.9 · 10 9 4.9 · 10 6 3 [Abu 3,11 ]SFTI-1 [6,7] 4.6 · 10 9 1.8 · 10 6 4 [Phe 5 ]SFTI-1 [10] 2.0 · 10 9 5 [KK]BiSFTI-1 (4.4 ± 0.4) · 10 8 (1.6 ± 0.2) · 10 8 6 [FF]BiSFTI-1 7 [KF]BiSFTI-1 (2.6 ± 0.2) · 10 8 (8.7 ± 0.2) · 10 8 (1.2 ± 0.2) · 10 10 (Ch) (5.3 ± 0.2) · 10 9 (T) 8 [FK]BiSFTI-1 ND (3.0 ± 0.4) · 10 8 (1.3 ± 0.3) · 10 9 (T) a With the exception of wild-type SFTI-1, all inhibitors are monocyclic with one disulfide bridge only or a head-to-tail cyclization (compound 3). A. Łe˛ gowska et al. An example of peptide splicing FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS 2353 chymotrypsin and monocyclic (disulfide bridge only) [Phe 5 ]SFTI-1. It is worth emphasizing that the con- ditions for the enzyme–inhibitor incubation used in the MS study differed from those applied for the determination of K a . To detect the complexes, we had to exchange the buffer for a more volatile one (an A B Fig. 2. MS spectra and results of HPLC analysis of (A) [FK]BiSFTI-1 (8) and (B) a mixture of b-trypsin and [FK]BiSFTI-1: peak 2, analogue 8 without tripeptide Abu-Thr-Lys; peak 3, analogue 8 with cleaved Abu-Thr-Lys and Gly-Arg fragments. An example of peptide splicing A. Łe˛ gowska et al. 2354 FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS 2333.2753 10+ 2592.4874 9+ 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z 0 1 2 3 4 5 Intens. x10 7 2333.2753 10+ 2592.4874 9+ 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 m/ z 0 1 2 3 4 5 Intens .x10 7 2486.4686 10+ 2762.4797 9+ Trypsin SFTI-1-trypsin complex A B Fig. 3. MS spectra of (A) bovine b-trypsin and (B) a mixture of b-trypsin and [KK]BiSFTI-1 (5). 2294.1491 11+ 2523.5201 10+ 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z 0.0 0.2 0.4 0.6 0.8 1.0 Intens. x10 8 2435.0841 11+ 2678.6160 10+ 2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z 0.0 0.5 1.0 1.5 2.0 Intens. x10 8 [Phe ]SFTI-1- chymotrypsin complex 5 A B Fig. 4. MS spectra of (A) bovine a-chymotrypsin and (B) a mixture of a-chymotrypsin and [FF]BiSFTI-1 (6). A. Łe˛ gowska et al. An example of peptide splicing FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS 2355 ammonium formate buffer, pH 5.8). In a separate experiment, we demonstrated that, under the conditions used for the MS study, the peptides exhibited their full inhibitory activity. In a similar way, we studied interac- tions of the remaining inhibitors, [KF]BiSFTI-1 (7) and [FK]BiSFTI-1 (8), by using ESI-MS. Compound 7 incubated with trypsin generated a peak corresponding to a noncovalent complex of SFTI-1 with trypsin. However, its formation was significantly slower com- pared to that of a mixture of 5 with trypsin. In a mix- ture of 8 with trypsin, only trace amounts of a complex of [Phe 5 ]SFTI-1 with the enzyme were found after 1 h of incubation. Compound 8 incubated with chymotryp- sin formed a noncovalent complex, although the reac- tion was definitely slower than that with compound 6. In a mixture of 7 with chymotrypsin, no noncovalent complex was found after 1 h of incubation. On the other hand, incubation of both compounds 7 and 8 in a mixture of trypsin and chymotrypsin resulted in the immediate high-yield formation of noncovalent com- plexes, SFTI-1–trypsin and [Phe 5 ]SFTI-1–chymotryp- sin, respectively. Figures 5 and 6 show the results of the MS analyses of those mixtures. These results clearly indicate that all dimeric ana- logues undergo proteolysis when incubated with target enzymes. In all cases, P 1 -P 1 ¢ reactive sites are located between positions 5 ⁄ 6 and 19 ⁄ 20 to release fragments with Ser6 and Lys19 at their N- and C-termini, respec- tively. The cleavage is followed by resynthesis of the peptide bond between Lys5 and Ser20 to pro- duce monocyclic SFTI-1 or its [Phe 5 ]SFTI-1 analogue. Figure 7 shows the splicing of the permutants medi- ated by target enzymes. These results are compatible with the inhibitory activity of the peptides (Table 1), with all of them being potent inhibitors of the target enzymes. Proteolytic susceptibility of the inhibitors was found to be in excellent agreement with their inhibitory activ- ity. In all cases where the dimeric species were less pro- teolytically resistant than their monocyclic reference compounds (i.e. SFTI-1 and [Phe 5 ]SFTI-1), their inhib- itory activities, expressed in terms of K a , were one order of magnitude lower. On the other hand, in all cases where the reference inhibitors were formed after proteolysis, the K a values matched those determined for the reference monomers. 10+ 2333.2814 10+ 2486.3689 10+ 2523.4314 9+ 2592.4929 2200 2300 2400 2500 2600 2700 2800 m/ z 0 2 4 6 8 Intens. x10 7 Trypsin Chymotrypsin SFTI-1-trypsin complex Fig. 5. MS spectrum of a mixture a-chymotrypsin, b-trypsin and [KF]BiSFTI-1 (7). An example of peptide splicing A. Łe˛ gowska et al. 2356 FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS Despite our expectations, the peptides did not form 2 : 1 complexes with the enzymes, nor did they simultaneously and independently (7 and 8) inhibit experimental proteinases; instead, they undergo prote- olysis. The enzymatic process involving proteolytic cleavage, combined with resynthesis of the peptide bond, is an intriguing finding. It may serve as a model for the in vivo formation of cyclic peptides by enzy- matic processing of their precursors generated by stan- dard translation. Some bioactive cyclic peptides Fig. 7. Splicing of the double-sequence SFTI-1 analogues mediated by target enzymes. 10+ 2333.2816 11+ 2435.0840 9+ 2592.4965 10+ 2678.6092 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z 0.0 0.2 0.4 0.6 0.8 1.0 Intens. x10 8 Trypsin [Phe 5 ]SFTI-1-chymotrypsin complex Fig. 6. MS spectrum of a mixture b-trypsin, a-chymotrypsin and [FK]BiSFTI-1 (8). A. Łe˛ gowska et al. An example of peptide splicing FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS 2357 comprising proteinogenic l-amino acids (e.g. the pep- tides produced by Caryophyllaceae plants) are likely to be formed by this mechanism, which can be named ‘peptide splicing’. Materials and methods Peptide synthesis All peptides were synthesized manually by the solid-phase method using standard Fmoc chemistry on 2-chlorotrityl chloride resin (substitution of Cl 1.46 meqÆg )1 ) (Calbio- chem-Novabiochem AG, La ¨ ufelfingen, Switzerland) apply- ing a previously described procedure [10]. During the last step, disulfide bridge formation was performed using 0.1 m solution of I 2 in MeOH as described previously [13]. All synthetic steps were monitored by HPLC analysis using an RP Kromasil-100, C 8 ,5lm column (4.6 · 250 mm) (Knauer, Berlin, Germany). The solvent systems were 0.1% trifluoroacetic acid (A) and 80% acetonitrile in A (B). A linear gradient of 20–80% B for 30 min was employed with a flow rate of 1 mlÆmin )1 , monitored at 226 nm. Finally, all peptides were purified on a semi-preparative HPLC column RP Kromasil-100, C 8 ,5lm column (8 · 250 mm) (Knauer) using the same solvent system as above. A linear gradient of 20–80% B for 30 min was employed with a flow rate of 2.5 mlÆmin )1 , monitored at 226 nm. To confirm the correct- ness of molecular weights of the peptides, MS analysis was carried out on a MALDI MS (Biflex III MALDI-TOF spectrometer; Bruker Daltonics) using a-CCA matrix. Determination of association constants The association constants were measured using a method developed in the laboratory of Laskowski et al. [14,15]. The procedure was described in detail previously [10]. The measurements were carried out at initial enzyme con- centrations over the ranges 5.1–5.8 nm and 2.8–7.2 nm for trypsin and chymotrypsin, respectively. To determine the K a values for [KF]BiSFTI-1 (7) and [FK]BiSFTI-1 (8) with trypsin in the presence of chymotrypsin, a two-fold molar excess over the inhibitor of the second enzyme was added to each of the experimental cuvettes, followed 5 min later by the addition of appropriate volumes of the trypsin and substrate solutions. The chymotrypsin inhibitory activity in the presence of trypsin was determined by the same proce- dure, using a reverse order of enzyme addition for preincu- bation. Proteolytic susceptibility assays Dimeric analogues of SFTI-1 were incubated in a 100 mm Tris-HCl buffer (pH 8.3) containing 20 mm CaCl 2 and 0.005% Triton X-100, using catalytic amounts of bovine b-trypsin or bovine a-chymotrypsin (1 mol%) [11]. The incubation was carried out at room temperature and aliquots of the mixture were taken out periodically and submitted to RP-HPLC analysis. Analysis of enzyme–inhibitor complexes using MS A 1.4 · 10 )5 m solution containing a proteolytic enzyme (trypsin or chymotrypsin) and inhibitor (1.7 · 10 )5 m)ina 20 mm aqueous ammonium formate buffer (pH 5.8) was incubated for predetermined periods of 0.5, 1 and 20 h. After incubation, the mixture was analysed directly by ESI- MS spectrometry. The experiments were performed using an FT-MS instrument (Apex-Ultra 7T; Bruker Daltonic) equipped with a dual ESI-MADI Apollo source (Agilent Technologies Inc., Santa Clara, CA, USA). The samples were infused at a flow rate of 2 llÆmin )1 . The potential between the spray needle and the orifice was set at 4.5 kV. Capillary temperature was 200 °C, and N 2 was used as a nebulizing gas. Acknowledgements This work was supported by the Ministry of Science and Higher Education (grant no. 2889 ⁄ H03 ⁄ 2008 ⁄ 34). References 1 Luckett RS, Garcia JJ, Barker AV, Konarev PR, Shewry AR, Clarke RL & Brady RL (1999) High- resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds. J Mol Biol 290, 525–533. 2 Marx UC, Korsinczky MLJ, Schirra HJ, Jones A, Condie B, Otvos L Jr & Craik DJ (2003) Enzymatic cyclization of a potent Bowman–Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide. J Biol Chem 278, 21782– 21789. 3 Qi R-F & Chi C (2005) Structural features and molecu- lar evolution of Bowman–Birk protease inhibitors and their potential application. Acta Biochim Biophys Sin 37, 283–292. 4 McBride JD, Watson EM, Brauer ABE, Jaulent AM & Leatherbarrow RJ (2002) Peptide mimics of the Bowman–Birk inhibitor reactive site loop. Biopolymers 66, 79–92. 5 Korsinczky MLJ, Schirra HJ & Craik DJ (2004) Sunflower trypsin inhibitor-1. Curr Protein Pept Sci 5, 351–364. 6 Zabłotna E, Kaz´ mierczak K, Jas ´ kiewicz A, Stawikowski M, Kupryszewski G & Rolka K (2002) Chemical synthesis and kinetic study of the smallest naturally An example of peptide splicing A. Łe˛ gowska et al. 2358 FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS occurring trypsin inhibitor SFTI-1 isolated from sun- flower seeds and its analogues. Biochem Biophys Res Commun 292, 855–859. 7 Zabłotna E, Kaz´ mierczak K, Jas ´ kiewicz A, Kupryszew- ski G & Rolka K (2002) Inhibition of bovine a-chymo- trypsin by cyclic trypsin inhibitor SFTI-1 isolated from sunflower seeds and its two acyclic analogues. LIPS 9, 131–134. 8 Korsinczky ML, Clark RJ & Craik DJ (2005) Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1. Biochemistry 44, 1145–1153. 9 Korsinczky ML, Schirra HJ, Rosengren KJ, West J, Condie BA, Otvos L, Anderson MA & Craik DJ (2001) Solution structures by 1 H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant. J Mol Biol 311, 579–591. 10 Łe˛gowska A, De˛bowski D, Lesner A, Wysocka M & Rolka K (2009) Introduction of non-natural amino acid residues into the substrate-specific P 1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors. Bioorg Med Chem 17, 3302– 3307. 11 Jaulent AM & Leatherbarrow RJ (2004) Design, synthesis and analysis of novel bicyclic and bifunc- tional protease inhibitors. Protein Eng Des Sel 17, 681–687. 12 Nesatyy VJ (2001) Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry. J Mass Spectrom 36, 950–959. 13 Yang Y, Sweeney WV, Schneider K, Chait BT & Tam JP (1994) Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX. Protein Sci 3, 1267–1275. 14 Empie MW & Laskowski M Jr (1982) Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor inter- actions with serine proteinases. Biochemistry 21, 2274– 2284. 15 Park SJ (1985) Effect of amino acid replacement in ovomucoid third domains upon their association with serine proteinases. PhD Thesis, Purdue University, IN, USA. A. Łe˛ gowska et al. An example of peptide splicing FEBS Journal 277 (2010) 2351–2359 ª 2010 The Authors Journal compilation ª 2010 FEBS 2359 . Inhibitory activity of double-sequence analogues of trypsin inhibitor SFTI-1 from sunflower seeds: an example of peptide splicing Anna Łe˛ gowska 1 , Adam Lesner 1 ,El _ zbieta Bulak 1 , Anna. x10 8 [Phe ]SFTI-1- chymotrypsin complex 5 A B Fig. 4. MS spectra of (A) bovine a-chymotrypsin and (B) a mixture of a-chymotrypsin and [FF]BiSFTI-1 (6). A. Łe˛ gowska et al. An example of peptide splicing FEBS. m/ z 0 2 4 6 8 Intens. x10 7 Trypsin Chymotrypsin SFTI-1 -trypsin complex Fig. 5. MS spectrum of a mixture a-chymotrypsin, b -trypsin and [KF]BiSFTI-1 (7). An example of peptide splicing A. Łe˛ gowska