NMR and SAXS characterization of the denatured state of the chemotactic protein CheY: Implications for protein folding initiation

The denatured state of a double mutant of the chemotactic protein CheY (F14 N/V83T) has been analyzed in the presence of 5 M urea, using small angle X- ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies sh ow that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexibl e links. A comparison of the contour length obtained for the chain at 5 M u rea with the one expected for a fully expanded chain suggests that similar to 25% of the residues are involved in residual structures. Conformational shifts of the or-protons, heteronuclear 15N-{H-1} NOEs and N-15 relaxation properties have been used to identify some regions in the protein that devi ate from a random coil behavior. According to these NMR data, the protein c an be divided into two subdomains, which largely coincide with the two fold ing subunits identified in a previous kinetic study of the folding of the p rotein. The first of these subdomains, spanning residues 1-70, is shown her e 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 r andom coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone H-1 chemical shifts were compared t o those in the intact protein under identical denaturing conditions. For th e region located in the first subdomain, this comparison shows that the obs erved 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 th e conclusions obtained by applying the protein engineering method to the ch aracterization of the folding reaction transition state.