The 1H, 13C, 15N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

Douglas V. Laurents, Marta Bruix, M. Angeles Jiménez, Jorge Santoro, Ester Boix, Mohammed Moussaoui, Maria Victoria Nogués, Manuel Rico

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Abstract

Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECP's ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all 1H, 15N, and backbone 13C resonances, to solve the 3D structure in aqueous solution and to quantify its residue-level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well-defined (backbone RMSD = 0.81 Å). The N-terminus and the loop composed of residues 114-123 are relatively well-ordered; in contrast, conformational diversity is observed for the loop segments 17-22, 65-68, and 92-95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two β-strands, and curiously, the first α-helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECP's unfolding thermodynamics. © 2009 Wiley Periodicals, Inc.
Original languageEnglish
Pages (from-to)1018-1028
JournalBiopolymers - Peptide Science Section
Volume91
DOIs
Publication statusPublished - 16 Dec 2009

Keywords

  • 3D-solution structure
  • Conformational stability
  • ECP cytotoxicity-structure relationship
  • ECP ribonuclease activity
  • Local and global unfoldings
  • NMR resonance assignment

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    Laurents, D. V., Bruix, M., Jiménez, M. A., Santoro, J., Boix, E., Moussaoui, M., Nogués, M. V., & Rico, M. (2009). The 1H, 13C, 15N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy. Biopolymers - Peptide Science Section, 91, 1018-1028. https://doi.org/10.1002/bip.21152