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The 1 H, 13 C, 15 N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy
Author(s) -
Laurents Douglas V.,
Bruix Marta,
Jiménez M. Angeles,
Santoro Jorge,
Boix Ester,
Moussaoui Mohammed,
Nogués Maria Victoria,
Rico Manuel
Publication year - 2009
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21152
Subject(s) - chemistry , rnase p , nuclear magnetic resonance spectroscopy , crystallography , cationic polymerization , residue (chemistry) , eosinophil cationic protein , protein structure , stereochemistry , rna , organic chemistry , eosinophil , biochemistry , medicine , asthma , gene
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 1 H, 15 N, and backbone 13 C 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. Biopolymers 91: 1018–1028, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com