Skip to main content
SpringerLink
Log in

Computational Prediction of New Intein Split Sites

  • Protocol
  • First Online:
Split Inteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1495))

Abstract

Split inteins have emerged as a powerful tool in protein engineering. We describe a reliable in silico method to predict viable split sites for the design of new split inteins. A computational circular permutation (CP) prediction method facilitates the search for internal permissive sites to create artificial circular permutants. In this procedure, the original amino- and carboxyl-termini are connected and new termini are created. The identified new terminal sites are promising candidates for the generation of new split sites with the backbone opening being tolerated by the structural scaffold. Here we show how to integrate the online usage of the CP predictor, CPred, in the search of new split intein sites.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Aranko AS, Wlodawer A, Iwai H (2014) Nature’s recipe for splitting inteins. Protein Eng Des Sel 27:263–271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5:446–461

    Article  CAS  PubMed  Google Scholar 

  3. Iwai H, Zuger S, Jin J, Tam PH (2006) Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett 580:1853–1858

    Article  CAS  PubMed  Google Scholar 

  4. Wu H, Hu Z, Liu XQ (1998) Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A 95:9226–9231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Muralidharan V, Muir TW (2006) Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat Methods 3:429–438

    Article  CAS  PubMed  Google Scholar 

  6. Volkmann G, Iwai H (2010) Protein trans-splicing and its use in structural biology: opportunities and limitations. Mol Biosyst 6:2110–2121

    Article  CAS  PubMed  Google Scholar 

  7. Zuger S, Iwai H (2005) Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies. Nat Biotechnol 23:736–740

    Article  CAS  PubMed  Google Scholar 

  8. Sun W, Yang J, Liu XQ (2004) Synthetic two-piece and three-piece split inteins for protein trans-splicing. J Biol Chem 279:35281–35286

    Article  CAS  PubMed  Google Scholar 

  9. Aranko AS, Zuger S, Buchinger E, Iwai H (2009) In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins. PLoS One 4:e5185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ludwig C, Schwarzer D, Zettler J, Garbe D, Janning P, Czeslik C, Mootz HD (2009) Semisynthesis of proteins using split inteins. Methods Enzymol 462:77–96

    Article  CAS  PubMed  Google Scholar 

  11. Mootz HD (2009) Split inteins as versatile tools for protein semisynthesis. Chembiochem 10:2579–2589

    Article  CAS  PubMed  Google Scholar 

  12. Lee YT, Su TH, Lo WC, Lyu PC, Sue SC (2012) Circular permutation prediction reveals a viable backbone disconnection for split proteins: an approach enabling identification of a new functional two-piece intein for protein trans splicing. PLoS One 7:e43820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tsai LC, Shyur LF, Lee SH, Lin SS, Yuan HS (2003) Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes. J Mol Biol 330:607–620

    Article  CAS  PubMed  Google Scholar 

  14. Ribeiro EA Jr, Ramos CH (2005) Circular permutation and deletion studies of myoglobin indicate that the correct position of its N-terminus is required for native stability and solubility but not for native-like heme binding and folding. Biochemistry 44:4699–4709

    Article  CAS  PubMed  Google Scholar 

  15. Lo WC, Lyu PC (2008) CPSARST: an efficient circular permutation search tool applied to the detection of novel protein structural relationships. Genome Biol 9:R11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lindqvist Y, Schneider G (1997) Circular permutations of natural protein sequences: structural evidence. Curr Opin Struct Biol 7:422–427

    Article  CAS  PubMed  Google Scholar 

  17. Vogel C, Morea V (2006) Duplication, divergence and formation of novel protein topologies. Bioessays 28:973–978

    Article  CAS  PubMed  Google Scholar 

  18. Qian Z, Lutz S (2005) Improving the catalytic activity of Candida antarctica lipase B by circular permutation. J Am Chem Soc 127:13466–13467

    Article  CAS  PubMed  Google Scholar 

  19. Todd AE, Orengo CA, Thornton JM (2002) Plasticity of enzyme active sites. Trends Biochem Sci 27:419–426

    Article  CAS  PubMed  Google Scholar 

  20. Li L, Shakhnovich EI (2001) Different circular permutations produced different folding nuclei in proteins: a computational study. J Mol Biol 306:121–132

    Article  CAS  PubMed  Google Scholar 

  21. Chen J, Wang J, Wang W (2004) Transition states for folding of circular-permuted proteins. Proteins 57:153–171

    Article  CAS  PubMed  Google Scholar 

  22. Bulaj G, Koehn RE, Goldenberg DP (2004) Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation. Protein Sci 13:1182–1196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cunningham BA, Hemperly JJ, Hopp TP, Edelman GM (1979) Favin versus concanavalin A: circularly permuted amino acid sequences. Proc Natl Acad Sci U S A 76:3218–3222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lo WC, Huang PJ, Chang CH, Lyu PC (2007) Protein structural similarity search by Ramachandran codes. BMC Bioinformatics 8:307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lo WC, Lee CC, Lee CY, Lyu PC (2009) CPDB: a database of circular permutation in proteins. Nucleic Acids Res 37:D328–D332

    Article  CAS  PubMed  Google Scholar 

  26. Lo WC, Wang LF, Liu YY, Dai T, Hwang JK, Lyu PC (2012) CPred: a web server for predicting viable circular permutations in proteins. Nucleic Acids Res 40:W232–W237

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Iwakura M, Nakamura T, Yamane C, Maki K (2000) Systematic circular permutation of an entire protein reveals essential folding elements. Nat Struct Biol 7:580–585

    Article  CAS  PubMed  Google Scholar 

  28. Paszkiewicz KH, Sternberg MJ, Lappe M (2006) Prediction of viable circular permutants using a graph theoretic approach. Bioinformatics 22:1353–1358

    Article  CAS  PubMed  Google Scholar 

  29. Amitai G, Shemesh A, Sitbon E, Shklar M, Netanely D, Venger I, Pietrokovski S (2004) Network analysis of protein structures identifies functional residues. J Mol Biol 344:1135–1146

    Article  CAS  PubMed  Google Scholar 

  30. Lo WC, Dai T, Liu YY, Wang LF, Hwang JK, Lyu PC (2012) Deciphering the preference and predicting the viability of circular permutations in proteins. PLoS One 7:e31791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shih CH, Huang SW, Yen SC, Lai YL, Yu SH, Hwang JK (2007) A simple way to compute protein dynamics without a mechanical model. Proteins 68:34–38

    Article  CAS  PubMed  Google Scholar 

  32. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577–2637

    Article  CAS  PubMed  Google Scholar 

  33. Panchenko AR, Madej T (2005) Structural similarity of loops in protein families: toward the understanding of protein evolution. BMC Evol Biol 5:10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Crasto CJ, Feng J (2001) Sequence codes for extended conformation: a neighbor-dependent sequence analysis of loops in proteins. Proteins 42:399–413

    Article  CAS  PubMed  Google Scholar 

  35. Lyu PC, Liff MI, Marky LA, Kallenbach NR (1990) Side chain contributions to the stability of alpha-helical structure in peptides. Science 250:669–673

    Article  CAS  PubMed  Google Scholar 

  36. Chakrabartty A, Kortemme T, Baldwin RL (1994) Helix propensities of the amino acids measured in alanine-based peptides without helix-stabilizing side-chain interactions. Protein Sci 3:843–852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Moreau RJ, Schubert CR, Nasr KA, Torok M, Miller JS, Kennedy RJ, Kemp DS (2009) Context-independent, temperature-dependent helical propensities for amino acid residues. J Am Chem Soc 131:13107–13116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lee B, Richards FM (1971) The interpretation of protein structures: estimation of static accessibility. J Mol Biol 55:379–400

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

This work was supported by Ministry of Science and Technology (MOST), Taiwan (101-2311-B-009-006-MY2, 102-2113-M-007-014, and 103-2113-M-007-016) and National Tsing Hua University (104N2051E1).

Author information

Authors and Affiliations

  1. Institute of Bioinformatics and Structural Biology, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, 30013, Hsinchu, Taiwan

    Yi-Zong Lee & Shih-Che Sue

  2. Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan

    Wei-Cheng Lo

  3. Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, Taiwan

    Wei-Cheng Lo

  4. Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan

    Shih-Che Sue

Authors
  1. Yi-Zong Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei-Cheng Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shih-Che Sue
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wei-Cheng Lo or Shih-Che Sue .

Editor information

Editors and Affiliations

  1. Institute of Biochemistry Department of Chemistry and Pharmacy, University of Muenster, Münster, Germany

    Henning D. Mootz

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Lee, YZ., Lo, WC., Sue, SC. (2017). Computational Prediction of New Intein Split Sites. In: Mootz, H. (eds) Split Inteins. Methods in Molecular Biology, vol 1495. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6451-2_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6451-2_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6449-9

  • Online ISBN: 978-1-4939-6451-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation