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

Abstract

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 approximately 25% of the residues are involved in residual structures. Conformational shifts of the alpha-protons, heteronuclear (15)N-[(1)H] NOEs and (15)N 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 (1)H 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.

Fig. 1.

Ribbon diagram of native CheY three-dimensional structure. Residues replaced in the mutant studied in the present work (F14N/V83T) are illustrated by their side chains. The two regions that show nonrandom coil behavior in the denatured protein (from residue 8 to 22 and 70 to 88) are represented in black. The diagram was produced using the program MOLSCRIPT (Kraulis 1991).

Fig. 2.

Differences of H^α chemical shifts (ppm) between F14N/V83T mutant and wild-type CheY. Data are obtained under identical conditions (pH 7, 25°C).

Fig. 3.

(A) Kratky plots, I(Q)Q² vs. Q, of mutant F14N/V83T in native conditions (dashed line) and of mutant F14N/V83T in 5 M urea (solid line). Each curve was normalized by the I(0) value obtained from the Guinier or Debye analysis. Both lines are the results of smooth fitting of the experimental data. (B) Experimental SAXS data (I(Q) vs. Q) for CheY in 5M urea. The solid line is the result of the fitting with equation (2).

Fig. 4.

¹⁵N-¹H HSQC spectrum of mutant F14N/V83T denatured in 5 M Urea, at pH 7 and 25°C. Peaks are labeled with the residue number. Nonassigned peaks are labeled with a question mark. Signals associated with an identified amino-acid type but not sequentially assigned are labeled with the letter corresponding to the amino-acid type.

Fig. 5.

H^α chemical shifts deviation from random coil values (Δδ = δ_experim. − δ_random-coil) as a function of protein sequence. (A) Entire mutant F14N/V83T; (B) isolated peptides spanning regions 5–30 and 62–89 of the mutant. Experimental values are obtained for mutant F14N/V83T and peptides under 5 M urea. Random coil values are from Wishart et al. 1995. Regions showing an overall trend to be in a nonrandom conformation in the mutant and conserving this tendency in the peptides are displayed with solid circles. Regions loosing this trend in isolated peptides are displayed with dashed circles. The secondary structure in the native protein is also shown.

Fig. 6.

Relaxation data of backbone amide ¹⁵N in mutant F14N/V83T denatured in 5 M urea plotted as a function of protein sequence. R₁: Longitudinal relaxation rates; R₂: Transverse relaxation rates. Heteronuclear ¹⁵N-¹H NOEs of backbone amide groups.

Fig. 7.

Calculated values of the reduced spectral density functions of residues as a function of protein sequence for CheY mutant F14N/V83T denatured in 5 M urea. J(0), J(ωN) and J(ωH) are the spectral densities at frequencies 0, 60 and 540 MHz, respectively. Dashed line represents the mean J(0) value calculated for residues 1–70, < J(0)_1–70 > (excluding residues that show highest J(0) values: 9–17 and 30–36). Dotted line represents the mean J(0) value calculated for residues 71–129, < J(0)_71–129 > (excluding residues that show highest J(0) values: 115–117).