title: Practical applications of hydrostatic pressure to refold proteins from inclusion bodies for NMR structural studies.
abstract: Recently, the hydrostatic pressure refolding method was reported as a practical tool for solubilizing and refolding proteins from inclusion bodies; however, there have been only a few applications for protein structural studies. Here, we report the successful applications of the hydrostatic pressure refolding method to refold proteins, including the MOE-2 tandem zinc-finger, the p62 PB1 domain, the GCN2 RWD domain, and the mTOR FRB domain. Moreover, the absence of aggregation and the correct folding of solubilized protein samples were evaluated with size exclusion chromatography and NMR experiments. The analyses of NMR spectra for MOE-2 tandem zinc-finger and GCN2 RWD further led to the determination of tertiary structures, which are consistent with those from soluble fractions. Overall, our results indicate that the hydrostatic pressure method is effective for preparing samples for NMR structural studies.
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Kita 21 Nishi 11, Kita, Sapporo 001-0021, Japan. k-ogura@mail.sci.hokudai.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Ogura
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kobashigawa
authors.forename: Tomohide
authors.initials: T
authors.lastname: Saio
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kumeta
authors.forename: Shinnosuke
authors.initials: S
authors.lastname: Torikai
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006874:Hydrostatic Pressure; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D011506:Proteins; D011994:Recombinant Proteins
keywords: NMR; hydrostatic pressure; protein structure; refolding