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Synlett 2017; 28(15): 1901-1906
DOI: 10.1055/s-0036-1588840
DOI: 10.1055/s-0036-1588840
cluster
Divergent Protein Synthesis of Bowman–Birk Protease Inhibitors, their Hydrodynamic Behavior and Co-crystallization with α-Chymotrypsin
Further Information
Publication History
Received: 28 March 2017
Accepted after revision: 30 April 2017
Publication Date:
24 May 2017 (online)
Published as part of the Cluster Recent Advances in Protein and Peptide Synthesis
Abstract
A divergent protein synthesis strategy was executed to effectively synthesize Bowman–Birk protease inhibitor (BBI) analogues using native chemical ligation of peptide hydrazides. Grafting selected residues from a potent trypsin inhibitor, sunflower trypsin inhibitor-1, onto the α-chymotrypsin-binding loop of BBI, resulted in a fourfold improvement of α-chymotrypsin inhibition. The crystal structure of a synthetic BBI analogue co-crystallized with α-chymotrypsin confirmed the correct protein fold and showed a similar overall structure to unmodified BBI in complex with α-chymotrypsin. Dynamic light scattering showed that C-terminal truncation of BBI led to increased self-association.
Key words
protein synthesis - divergent synthesis - Bowman–Birk protease inhibitor - X-ray crystallography - α-chymotrypsin - native chemical ligationSupporting Information
- Experimental details, compound characterization, enzyme inhibition and DLS measurements. Crystal structure coordinates and structure factors have been deposited in PDB entries 5J4Q (BBI (1):α-chymotrypsin) and 5J4S (11:α-chymotrypsin).Supporting information for this article is available online at https://doi.org/10.1055/s-0036-1588840.
- Supporting Information
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References and Notes
- 1 Scott DE. Bayly AR. Abell C. Skidmore J. Nat. Rev. Drug Discov. 2016; 15: 533
- 2 Lindsley CW. Weaver D. Bridges TM. Kennedy JP. In Chemical Biology . John Wiley and Sons; 2012: 65
- 3 Edwards PJ. LaPlante SR. In Peptide Drug Discovery and Development . Wiley-VCH; Weinheim: 2011: 1
- 4 Miranda LP. Shao H. Williams J. Chen S.-Y. Kong T. Garcia R. Chinn Y. Fraud N. O'Dwyer B. Ye J. Wilken J. Low DE. Cagle EN. Carnevali M. Lee A. Song D. Kung A. Bradburne JA. Paliard X. Kochendoerfer GG. J. Am. Chem. Soc. 2007; 129: 13153
- 5a Qiu Y. Taichi M. Wei N. Yang H. Luo KQ. Tam JP. J. Med. Chem. 2017; 60: 504
- 5b Qu H. Smithies BJ. Durek T. Craik DJ. Aust. J. Chem. 2017; 70: 152
- 6 Leung D. Abbenante G. Fairlie DP. J. Med. Chem. 2000; 43: 305
- 7 Drag M. Salvesen GS. Nat. Rev. Drug Discov. 2010; 9: 690
- 8a Bowman DE. Experimental Biol. Med. 1946; 63: 547
- 8b Birk Y. Gertler A. Khalef S. Biochem. J. 1963; 87: 281
- 9a Ryan CA. Annu. Rev. Phytopathol. 1990; 28: 425
- 9b Habib H. Fazili KM. Biotechnol. Mol. Biol. Rev. 2007; 2: 68
- 10 Birk Y. Biochim. Biophys. Acta 1961; 54: 378
- 11a Birk Y. Gertler A. Khalef S. Biochim. Biophys. Acta 1967; 147: 402
- 11b Birk Y. Int. J. Pept. Protein Res. 1985; 25: 113
- 12a Malkowicz SB. McKenna WG. Vaughn DJ. Wan XS. Propert KJ. Rockwell K. Marks SH. F. Wein AJ. Kennedy AR. Prostate 2001; 48: 16
- 12b Lin LL. Mick R. Ware J. Metz J. Lustig R. Vapiwala N. Rengan R. Kennedy AR. Oncol. Lett. 2014; 7: 1151
- 13 Hernández-Ledesma B. Hsieh C.-C. de Lumen BO. Food Chem. 2009; 115: 574
- 14 Odani S. Ono T. J. Biochem. 1980; 88: 1555
- 15 Jaulent AM. Leatherbarrow RJ. Protein Eng., Des. Sel. 2004; 17: 681
- 16 Fang G.-M. Li Y.-M. Shen F. Huang Y.-C. Li J.-B. Lin Y. Cui H.-K. Liu L. Angew. Chem. Int. Ed. 2011; 50: 7645
- 17 Dawson P. Muir T. Clark-Lewis I. Kent S. Science 1994; 266: 776
- 18 Native chemical ligation was achieved by dissolution of peptide hydrazide in a mixture of 90:10 0.2 M Na2HPO4/6 M Gu·HCl and MeCN (pH adjusted to 3.0) at 0° C and addition of NaNO2 (5 equiv). After 20 min, MESNa (50 equiv) and Cys-peptide (1.1 equiv) were added and pH was adjusted to 7.0. Excess 1,4-dithiothreitol was added after 16 h, and the product was purified by RP-HPLC.
- 19 Protein folding was achieved by dissolution of unfolded protein in 6 mM Gu·HCl/2 mM mercaptoethanol/0.2 mM GSSG, 0.1 mM EDTA/80 mM Tris (pH 7.9) at 0.05 mg/mL.
- 20 UPLC and LC–MS were used to characterize the folded proteins. UPLC (C18 column, 2.1 mm × 150 mm, 95:5 to 5:95 water/MeCN + 0.1% TFA over 16 min). LC–MS (C18 column, 2.1 mm × 50 mm, 95:5 to 5:95 water/MeCN + 0.1% formic acid over 4 min). Protein 9: UPLC: t R 5.1 min, LC–MS: m/z calcd 7840.7; found m/4: 1961.0, m/5: 1569.0, m/6: 1307.5, m/7: 1121.0, m/8: 981.1. Protein 10: UPLC: t R 4.7 min, LC–MS: m/z calcd 7110.0; found m/4: 1778.2, m/5: 1422.7, m/6: 1186.0, m/7: 1016.7. Protein 11: UPLC: t R 5.2 min, LC–MS: m/z calcd 7880.7; found m/5: 1577.1, m/6: 1314.4, m/7: 1126.7, m/8: 985.9. Protein 12: UPLC: t R 5.2 min, LC–MS: m/z calcd 7150.0; found m/4: 1788.5, m/5: 1431.0, m/6: 1192.6, m/7: 1022.4, m/8: 894.5.
- 21a Fujinari EM. Courthaudon LO. J. Chromatogr. A 1992; 592: 209
- 21b Fujinari EM. In Developments in Food Science . Vol. 39. Wetzel D. Charalambous G. Elsevier; Amsterdam: 1998: 431
- 22 Appel W. Clin. Biochem. 1986; 19: 317
- 23a Zabłotna E. Jaśkiewicz A. Łęgowska A. Miecznikowska H. Lesner A. Rolka K. J. Pept. Sci. 2007; 13: 749
- 23b Debowski D. Łukajtis R. Filipowicz M. Strzelecka P. Wysocka M. Łęgowska A. Lesner A. Rolka K. Pept. Sci. 2013; 100: 154
- 24 Luckett S. Garcia RS. Barker JJ. Konarev AV. Shewry PR. Clarke AR. Brady RL. J. Mol. Biol. 1999; 290: 525
- 25 Hopkins AL. Groom CR. Alex A. Drug Discovery Today 2004; 9: 430
- 26 Korsinczky ML. J. Schirra HJ. Rosengren KJ. West J. Condie BA. Otvos L. Anderson MA. Craik DJ. J. Mol. Biol. 2001; 311: 579
- 27 Ando S. Yasutake A. Waki M. Nishino N. Kato T. Izumiya N. Biochim. Biophys. Acta 1987; 916: 527