title: Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility.
abstract: A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS(2)) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS(2)-tag is replaced with non-isotope labeled PrS(2)-tag, silencing the NMR signals from PrS(2)-tag in isotope-filtered (1)H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS(2)-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS(2) (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS(2)-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS(2)-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone (1)H, (15)N and (13)C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear (1)H-(15)N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kobayashi
authors.forename: G V T
authors.initials: GV
authors.lastname: Swapna
authors.forename: Kuen-Phon
authors.initials: KP
authors.lastname: Wu
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Afinogenova
authors.forename: Kenith
authors.initials: K
authors.lastname: Conover
authors.forename: Binchen
authors.initials: B
authors.lastname: Mao
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Masayori
authors.initials: M
authors.lastname: Inouye
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D017293:Protein S / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D017293:Protein S; D011506:Proteins; C092748:RbfA protein, E coli; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins