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Salvage or Recovery of Failed Targets by In Situ Proteolysis

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Book cover Structural Genomics and Drug Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1140))

Abstract

In situ proteolysis is the method of proactively adding tiny amounts of nonspecific proteases to aid in the crystallization of proteins and protein macromolecular complexes. The simplicity of the procedure and high recovery rate make it a method of first choice for recalcitrant targets. An improved and updated in situ proteolysis protocol used in high-throughput structural biology platforms is described.

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References

  1. Nouwen N, Stahlberg H, Pugsley AP, Engel A (2000) Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy. EMBO J 19:2229–2236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Longhi S, Ferron F, Egloff MP (2007) Protein engineering. Methods Mol Biol 363:59–89

    Article  CAS  PubMed  Google Scholar 

  3. Danley DE, Haggan ME, Cunningham D, Fennell KF, Pauly TA, LeMotte PK (2000) A crystallizable form of RIIbeta regulatory domain obtained by limited proteolysis. Acta Crystallogr D Biol Crystallogr 56:1038–1041

    Article  CAS  PubMed  Google Scholar 

  4. Cui J, Somerville RL (1993) The TyrR protein of Escherichia coli, analysis by limited proteolysis of domain structure and ligand-mediated conformational changes. J Biol Chem 268:5040–5047

    Article  CAS  PubMed  Google Scholar 

  5. Gao X, Bain K, Bonanno JB, Buchanan M, Henderson D, Lorimer D, Marsh C, Reynes JA, Sauder JM, Schwinn K et al (2005) High-throughput limited proteolysis/mass spectrometry for protein domain elucidation. J Struct Funct Genomics 6:129–134

    Article  CAS  PubMed  Google Scholar 

  6. Sumner JB, Howell SF (1936) The isolation of a fourth crystallizable jack bean globulin through the digestion of canavalin with trypsin. J Biol Chem 113:607–610

    Article  CAS  Google Scholar 

  7. Sawaya MR, Pelletier H, Kumar A, Wilson SH, Kraut J (1994) Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science 264:1930–1935

    Article  CAS  PubMed  Google Scholar 

  8. Campbell EA, Muzzin O, Chlenov M, Sun JL, Olson CA, Weinman O, Trester-Zedlitz ML, Darst SA (2002) Structure of the bacterial RNA polymerase promoter specificity sigma subunit. Mol Cell 9:527–539

    Article  CAS  PubMed  Google Scholar 

  9. Hoedemaeker FJ, Signorelli T, Johns K, Kuntz DA, Rose DR (1997) A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219. Design, expression, and crystal structure at 2.4 A resolution. J Biol Chem 272:29784–29789

    Article  CAS  PubMed  Google Scholar 

  10. Machius M, Wiegand G, Huber R (1995) Crystal structure of calcium-depleted Bacillus licheniformis alpha-amylase at 2.2 A resolution. J Mol Biol 246:545–559

    Article  CAS  PubMed  Google Scholar 

  11. Nieves-Alicea R, Focia PJ, Craig SP III, Eakin AE (1998) Limited proteolysis of a trypanosomal hypoxanthine phosphoribosyltransferase yields crystals that diffract X-rays to near atomic resolution. Biochim Biophys Acta 1388:500–505

    Article  CAS  PubMed  Google Scholar 

  12. Umland TC, Taylor KL, Rhee S, Wickner RB, Davies DR (2001) The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p. Proc Natl Acad Sci U S A 98:1459–1464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mandel CR, Gebauer D, Zhang H, Tong L (2006) A serendipitous discovery that in situ proteolysis is essential for the crystallization of yeast CPSF-100 (Ydh1p). Acta Crystallogr F Struct Biol Cryst Commun 62:1041–1045

    Article  CAS  Google Scholar 

  14. Johnson S, Roversi P, Espina M, Deane JE, Birket S, Picking WD, Blocker A, Picking WL, Lea SM (2006) Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. Acta Crystallogr F Struct Biol Cryst Commun 62:865–868

    Article  CAS  Google Scholar 

  15. Bai Y, Auperin TC, Tong L (2007) The use of in situ proteolysis in the crystallization of murine CstF-77. Acta Crystallogr F Struct Biol Cryst Commun 63:135–138

    Article  CAS  Google Scholar 

  16. McPherson A (1990) Current approaches to macromolecular crystallization. Eur J Biochem 189:1–23

    Article  CAS  PubMed  Google Scholar 

  17. Gaur RK, Kupper MB, Fischer R, Hoffmann KM (2004) Preliminary X-ray analysis of a human V(H) fragment at 1.8 A resolution. Acta Crystallogr D Biol Crystallogr 60:965–967

    Article  PubMed  Google Scholar 

  18. Taneja B, Patel A, Slesarev A, Mondragon A (2006) Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases. EMBO J 25:398–408

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dong A, Xu X, Edwards AM, Midwest Center for Structural Genomics Structural Genomics Consortium (2007) In situ proteolysis for protein crystallization and structure determination. Nat Methods 4:1019–1021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wernimont A, Edwards A (2009) In situ proteolysis to generate crystals for structure determination: an update. PLoS ONE 4:e5094

    Article  PubMed  PubMed Central  Google Scholar 

  21. Little DJ, Whitney JC, Robinson H, Yip P, Nitz M, Howell PL (2012) Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB. Acta Crystallogr F Struct Biol Cryst Commun 68:842–845

    Article  CAS  Google Scholar 

  22. Gheyi T, Rodgers L, Romero R, Sauder JM, Burley SK (2010) Mass spectrometry guided in situ proteolysis to obtain crystals for X-ray structure determination. J Am Soc Mass Spectrom 21:1795–1801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Abskharon RN, Soror SH, Pardon E, El HH, Legname G, Steyaert J, Wohlkonig A (2011) Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes. Protein Eng Des Sel 24:737–741

    Article  CAS  PubMed  Google Scholar 

  24. Ismail SA, Chen YX, Miertzschke M, Vetter IR, Koerner C, Wittinghofer A (2012) Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119. EMBO J 31:4085–4094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kobayashi K, Saito K, Ishitani R, Ito K, Nureki O (2012) Structural basis for translation termination by archaeal RF1 and GTP-bound EF1alpha complex. Nucleic Acids Res 40:9319–9328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lee HR, Choi WC, Lee S, Hwang J, Hwang E, Guchhait K, Haas J, Toth Z, Jeon YH, Oh TK et al (2011) Bilateral inhibition of HAUSP deubiquitinase by a viral interferon regulatory factor protein. Nat Struct Mol Biol 18:1336–1344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yachnin BJ, Colin DY, Volpato JP, Ebert M, Pelletier JN, Berghuis AM (2011) Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure. Acta Crystallogr F Struct Biol Cryst Commun 67:1316–1322

    Article  CAS  Google Scholar 

  28. Fischer H, Polikarpov I, Craievich AF (2004) Average protein density is a molecular-weight-dependent function. Protein Sci 13:2825–2828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgment

We are grateful for members of SGC and MCSG for the domestication of the in situ proteolysis method on the high-throughput structural biology platforms.

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Authors and Affiliations

  1. Structural Genomics Consortium, University of Toronto, 101 College Street, MaRS Centre, South Tower, Suite 700, Toronto, ON, Canada, M5G 1L7

    Yufeng Tong

  2. Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, ON, Canada, M5G 1L7

    Aiping Dong & Amy Wernimont

  3. Midwest Center for Structural Genomics, University of Toronto, 112 College Street, Toronto, ON, Canada, M5G 1L6

    Xiaohui Xu

Authors
  1. Yufeng Tong
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  2. Aiping Dong
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  3. Xiaohui Xu
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  4. Amy Wernimont
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Corresponding author

Correspondence to Yufeng Tong .

Editor information

Editors and Affiliations

  1. Center for Structural Genomics of Infectious Diseases Midwest Center for Structural Genomics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Wayne F. Anderson

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Tong, Y., Dong, A., Xu, X., Wernimont, A. (2014). Salvage or Recovery of Failed Targets by In Situ Proteolysis. In: Anderson, W.F. (eds) Structural Genomics and Drug Discovery. Methods in Molecular Biology, vol 1140. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0354-2_14

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  • DOI: https://doi.org/10.1007/978-1-4939-0354-2_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-0353-5

  • Online ISBN: 978-1-4939-0354-2

  • eBook Packages: Springer Protocols

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