Register      Login
Australian Journal of Chemistry Australian Journal of Chemistry Society
An international journal for chemical science
Shopping Cart: ( empty )
You are here: Home > Journals > CH > CH14342
RESEARCH FRONT
Contents Vol 67(12)

Crystal ‘Unengineering’: Reducing the Crystallisability of Sulfolobus solfataricus Hjc

Claire L. Middleton A , Joanne L. Parker B , Gavin J. Knott C , Malcolm F. White D and Charles S. Bond C E
+ Author Affiliations
- Author Affiliations

A School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.

B Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

C School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia.

D Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, KY16 9ST, UK.

E Corresponding author: Email: charles.bond@uwa.edu.au

Australian Journal of Chemistry 67(12) 1818-1823 https://doi.org/10.1071/CH14342
Submitted: 29 May 2014  Accepted: 20 June 2014   Published: 18 August 2014

Abstract

The protein Hjc from the thermophilic archaeon Sulfolobus solfataricus (Ss) presented many challenges to both structure solution and formation of stable complexes with its substrate, the DNA four-way or Holliday junction. As the challenges were caused by an uncharacteristically high propensity for rapid and promiscuous crystallisation, we investigated the molecular cause of this behaviour, corrected it by mutagenesis, and solved the X-ray crystal structures of the two mutants. An active site mutant SsHjcA32A crystallised in space group I23 (a 144.2 Å; 68 % solvent), and a deletion of a key crystal contact site, SsHjcδ62–63 crystallised in space group P21 (a 64.60, b 61.83, c 55.25 Å; β = 95.74°; 28 % solvent). Characterisation and comparative analysis of the structures are presented along with discussion of the pitfalls of the use of protein engineering to alter crystallisability while maintaining biological function.


References

[1]  N. E. Chayen, Prog. Biophys. Mol. Biol. 2005, 88, 329.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXivVSitQ%3D%3D&md5=0aba8c2858932f61dabfd71dfd05c497CAS | 15652248PubMed |

[2]  J. Newman, Methods 2011, 55, 73.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtlaqt73L&md5=e09a56c0d298464d746866f03bbb9396CAS | 21571072PubMed |

[3]  M. Kvaratskhelia, M. F. White, J. Mol. Biol. 2000, 295, 193.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXht1CrsA%3D%3D&md5=6e794b47b214edd5e595b624a214f945CAS | 10623519PubMed |

[4]  L. Krejci, V. Altmannova, M. Spirek, X. Zhao, Nucleic Acids Res. 2012, 40, 5795.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhtFSlsLzE&md5=87338bb56c87108be57a2b5aaa9c0a35CAS | 22467216PubMed |

[5]  M. F. White, Biochem. Soc. Trans. 2011, 39, 15.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsVSitbk%3D&md5=bbc5b2e875af7ce6bff47b9c470ae5a7CAS | 21265740PubMed |

[6]  R. Holliday, Genetics 1964, 50, 323.
         | 1:CAS:528:DyaF2cXkslSqs7c%3D&md5=3aa21a7f270b4cdd58dbf29f240bf2a1CAS | 14207702PubMed |

[7]  D. M. Lilley, M. F. White, Nat. Rev. Mol. Cell Biol. 2001, 2, 433.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXks1ags7w%3D&md5=f0368619981b4cf11c03797562ec3d86CAS | 11389467PubMed |

[8]  D. R. Duckett, A. I. Murchie, M. J. Giraud-Panis, J. R. Pohler, D. M. Lilley, Philos. Trans. R. Soc., B 1995, 347, 27.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXltlGktbc%3D&md5=888b5e780d68492c626fc8ec87689cb2CAS |

[9]  M. Ortiz-Lombardia, A. Gonzalez, R. Eritja, J. Aymami, F. Azorin, M. Coll, Nat. Struct. Biol. 1999, 6, 913.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXmslCkt7c%3D&md5=622d7a43d252b3cacbe8c1e4ac7454afCAS | 10504723PubMed |

[10]  J. M. Hadden, M. A. Convery, A. C. Declais, D. M. Lilley, S. E. Phillips, Nat. Struct. Biol. 2001, 8, 62.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXjslyrtQ%3D%3D&md5=871c3246c173bcf4babd99e3ab5c3fc4CAS | 11135673PubMed |

[11]  H. Raaijmakers, O. Vix, I. Toro, S. Golz, B. Kemper, D. Suck, EMBO J. 1999, 18, 1447.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXisVWnsb4%3D&md5=bcce36f4e7d83157ea33bce180c60842CAS | 10075917PubMed |

[12]  M. Ariyoshi, D. G. Vassylyev, H. Iwasaki, H. Nakamura, H. Shinagawa, K. Morikawa, Cell 1994, 78, 1063.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXmsVOqtro%3D&md5=8a5e32e4167854a6774781bcb8cd5c38CAS | 7923356PubMed |

[13]  J. B. Rafferty, E. L. Bolt, T. A. Muranova, S. E. Sedelnikova, P. Leonard, A. Pasquo, P. J. Baker, D. W. Rice, G. J. Sharples, R. G. Lloyd, Structure 2003, 11, 1557.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXps1Ortb4%3D&md5=10176b2aaa0a58517b4d7a447daa38b2CAS | 14656440PubMed |

[14]  N. McGregor, S. Ayora, S. Sedelnikova, B. Carrasco, J. C. Alonso, P. Thaw, J. Rafferty, Structure 2005, 13, 1341.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXpvFentrY%3D&md5=2a9acac51b0471d31c48c6e818622ebfCAS | 16154091PubMed |

[15]  C. L. Middleton, J. L. Parker, D. J. Richard, M. F. White, C. S. Bond, Nucleic Acids Res. 2004, 32, 5442.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXovVOitLc%3D&md5=11cc1e4f896db03ee244ea8f59359f74CAS | 15479781PubMed |

[16]  C. L. Middleton, J. L. Parker, D. J. Richard, M. F. White, C. S. Bond, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 171.
         | Crossref | GoogleScholarGoogle Scholar |

[17]  C. S. Bond, M. Kvaratskhelia, D. Richard, M. F. White, W. N. Hunter, Proc. Natl. Acad. Sci. U.S.A 2001, 98, 5509.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXjs1Wgu70%3D&md5=c7152c6ed768f57b00b71f9518a90698CAS | 11331763PubMed |

[18]  T. Nishino, K. Komori, D. Tsuchiya, Y. Ishino, K. Morikawa, Structure 2001, 9, 197.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXisVOqsro%3D&md5=8fa373e82c5e2ad6fac6cb8618d9d719CAS | 11286886PubMed |

[19]  C. Biertümpfel, W. Yang, D. Suck, Nature 2007, 449, 616.
         | Crossref | GoogleScholarGoogle Scholar | 17873859PubMed |

[20]  J. M. Hadden, A. C. Declais, S. B. Carr, D. M. Lilley, S. E. Phillips, Nature 2007, 449, 621.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFagt7fE&md5=d9bb4357900ba090fc46e9f1f5ef3adbCAS | 17873858PubMed |

[21]  B. Heras, M. A. Edeling, K. A. Byriel, A. Jones, S. Raina, J. L. Martin, Structure 2003, 11, 139.
         | 1:CAS:528:DC%2BD3sXhtVamt7o%3D&md5=198dd2e4e4c2e63a79c22a760e4111d5CAS | 12575933PubMed |

[22]  M. Kvaratskhelia, B. N. Wardleworth, C. S. Bond, J. M. Fogg, D. M. Lilley, M. F. White, J. Biol. Chem. 2002, 277, 2992.
         | 1:CAS:528:DC%2BD38XhtVSgtb4%3D&md5=aa8cbe687a40b461e142fddc27ccf9a0CAS | 11709558PubMed |

[23]  M. Lee, D. M. Passon, S. Hennig, A. H. Fox, C. S. Bond, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 981.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsVCgurrJ&md5=a019a8b8ba5b7bd65c36c68fd266625cCAS |

[24]  S. A. Messing, S. B. Gabelli, Q. Liu, H. Celesnik, J. G. Belasco, S. A. Pineiro, L. M. Amzel, Structure 2009, 17, 472.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXivVKitrs%3D&md5=a32c59ac0d4a01276f424c70b7dd12e2CAS | 19278661PubMed |

[25]  M. Kvaratskhelia, M. F. White, J. Mol. Biol. 2000, 297, 923.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXhvFKnt70%3D&md5=43bcc5c7e18cd0b018c104a7775e4755CAS | 10736227PubMed |

[26]  B. W. Matthews, J. Mol. Biol. 1968, 33, 491.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaF1cXktVynurw%3D&md5=9cd5a6d8cf925c55948e4c62f8300305CAS | 5700707PubMed |

[27]  A. Vagin, A. Teplyakov, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2000, 56, 1622.
         | Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3M%2Fpt1KjsA%3D%3D&md5=274b0dcdbf498c63362e3400a5301077CAS |

[28]  Collaborative Computational Project, Number 4 Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760.
         | Crossref | GoogleScholarGoogle Scholar |

[29]  G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240.
         | Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2czpsFegsw%3D%3D&md5=65709a10de05d2e1b5e1336ee18c24baCAS |

[30]  O. S. Smart, T. O. Womack, C. Flensburg, P. Keller, W. Paciorek, A. Sharff, C. Vonrhein, G. Bricogne, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68, 368.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xlt1Gns78%3D&md5=003841db80896f900ff1a9afd9986921CAS |

[31]  E. Blanc, P. Roversi, C. Vonrhein, C. Flensburg, S. M. Lea, G. Bricogne, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2210.
         | Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2cnjs1SgsA%3D%3D&md5=79ead14d6cfb6fe0f4bca10da2bf7580CAS |

[32]  P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXksFKisb8%3D&md5=21208f5f13f477e014818cb1e41427b4CAS |

[33]  V. B. Chen, W. B. Arendall, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L. W. Murray, J. S. Richardson, D. C. Richardson, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 12.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXit1Kktg%3D%3D&md5=a8a63882e4c89734c861fc02f6da4286CAS |

[34]  C. S. Bond, A. W. Schuttelkopf, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 510.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXkvVykurc%3D&md5=b6d699a356fac11005c12c10e5f95310CAS |

[35]  C. S. Bond, J. Appl. Crystallogr. 2003, 36, 350.
         | Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXitlWitbg%3D&md5=a80d5790278314ae367c4f0c4c5defffCAS |