Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor.
Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of...
Guardado en:
| Autores principales: | , , , , , , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Public Library of Science (PLoS)
2014
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/ed72434ee1ae4e5c979266e2053710c7 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| id |
oai:doaj.org-article:ed72434ee1ae4e5c979266e2053710c7 |
|---|---|
| record_format |
dspace |
| spelling |
oai:doaj.org-article:ed72434ee1ae4e5c979266e2053710c72021-11-25T06:00:10ZAtomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor.1932-620310.1371/journal.pone.0106936https://doaj.org/article/ed72434ee1ae4e5c979266e2053710c72014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0106936https://doaj.org/toc/1932-6203Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and Rfree of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β2αβ fold characteristic for Kazal-family serine proteinase inhibitors.Edyta KoperaWojciech BalMartina Lenarčič ŽivkovičAngela DvornykBarbara KludkiewiczKrystyna GrzelakIgor ZhukovWłodzimierz Zagórski-OstojaMariusz JaskolskiSzymon KrzywdaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 9, p e106936 (2014) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Medicine R Science Q |
| spellingShingle |
Medicine R Science Q Edyta Kopera Wojciech Bal Martina Lenarčič Živkovič Angela Dvornyk Barbara Kludkiewicz Krystyna Grzelak Igor Zhukov Włodzimierz Zagórski-Ostoja Mariusz Jaskolski Szymon Krzywda Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor. |
| description |
Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and Rfree of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β2αβ fold characteristic for Kazal-family serine proteinase inhibitors. |
| format |
article |
| author |
Edyta Kopera Wojciech Bal Martina Lenarčič Živkovič Angela Dvornyk Barbara Kludkiewicz Krystyna Grzelak Igor Zhukov Włodzimierz Zagórski-Ostoja Mariusz Jaskolski Szymon Krzywda |
| author_facet |
Edyta Kopera Wojciech Bal Martina Lenarčič Živkovič Angela Dvornyk Barbara Kludkiewicz Krystyna Grzelak Igor Zhukov Włodzimierz Zagórski-Ostoja Mariusz Jaskolski Szymon Krzywda |
| author_sort |
Edyta Kopera |
| title |
Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor. |
| title_short |
Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor. |
| title_full |
Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor. |
| title_fullStr |
Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor. |
| title_full_unstemmed |
Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor. |
| title_sort |
atomic resolution structure of a protein prepared by non-enzymatic his-tag removal. crystallographic and nmr study of gmspi-2 inhibitor. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2014 |
| url |
https://doaj.org/article/ed72434ee1ae4e5c979266e2053710c7 |
| work_keys_str_mv |
AT edytakopera atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT wojciechbal atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT martinalenarciczivkovic atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT angeladvornyk atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT barbarakludkiewicz atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT krystynagrzelak atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT igorzhukov atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT włodzimierzzagorskiostoja atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT mariuszjaskolski atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor AT szymonkrzywda atomicresolutionstructureofaproteinpreparedbynonenzymatichistagremovalcrystallographicandnmrstudyofgmspi2inhibitor |
| _version_ |
1718414315121278976 |