Journal of Biomolecular NMR

, Volume 52, Issue 4, pp 303–313 | Cite as

Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

  • Hiroshi Kobayashi
  • G. V. T. Swapna
  • Kuen-Phon Wu
  • Yuliya Afinogenova
  • Kenith Conover
  • Binchen Mao
  • Gaetano T. Montelione
  • Masayori Inouye
Article

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 (PrS2) 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 PrS2-tag is replaced with non-isotope labeled PrS2-tag, silencing the NMR signals from PrS2-tag in isotope-filtered 1H-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 PrS2-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS2 (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS2-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS2-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 1H, 15N and 13C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear 1H–15N 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.

Keywords

Protein S Intein Protein ligation Protein NMR RbfA 

Supplementary material

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Hiroshi Kobayashi
    • 1
  • G. V. T. Swapna
    • 2
  • Kuen-Phon Wu
    • 1
  • Yuliya Afinogenova
    • 1
  • Kenith Conover
    • 2
  • Binchen Mao
    • 2
  • Gaetano T. Montelione
    • 1
    • 2
  • Masayori Inouye
    • 1
  1. 1.Department of Biochemistry, Robert Wood Johnson Medical SchoolCenter for Advanced Biotechnology and MedicinePiscatawayUSA
  2. 2.Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, RutgersThe State University of New JerseyPiscatawayUSA

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