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1 Instituto de Estructura de la Materia, CSIC, 28006 Madrid, Spain
2 European Molecular Biology Laboratory, Heidelberg, D-69012, Germany
3 LURE (CNRS/CEA/MENRT) B|fQt 209D, Université de Paris-Sud, Orsay, France
Reprint requests to: Dr. Marta Bruix, Instituto de Estructura de la Materia, CSIC, Serrano, 119, 28006 Madrid, Spain; e-mail: marta{at}malika.iem.csic.es; fax: 34 91 564 24 31.
The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that 
25% of the residues are involved in residual structures. Conformational shifts of the 
-protons, heteronuclear 15N-{1H} NOEs and 15N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1–70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone 1H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state.
Keywords: Denatured state; residual structures; folding initiation; heteronuclear NMR; backbone dynamics
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