Structural disorder of folded proteins: isotope-edited 2D IR spectroscopy and Markov state modeling

Abstract

The conformational heterogeneity of the N-terminal domain of the ribosomal protein L9 (NTL91-39) in its folded state is investigated using isotope-edited two-dimensional infrared spectroscopy. Backbone carbonyls are isotope-labeled ((13)C=(18)O) at five selected positions (V3, V9, V9G13, G16, and G24) to provide a set of localized spectroscopic probes of the structure and solvent exposure at these positions. Structural interpretation of the amide I line shapes is enabled by spectral simulations carried out on structures extracted from a recent Markov state model. The V3 label spectrum indicates that the β-sheet contacts between strands I and II are well folded with minimal disorder. The V9 and V9G13 label spectra, which directly probe the hydrogen-bond contacts across the β-turn, show significant disorder, indicating that molecular dynamics simulations tend to overstabilize ideally folded β-turn structures in NTL91-39. In addition, G24-label spectra provide evidence for a partially disordered α-helix backbone that participates in hydrogen bonding with the surrounding water.

Figure 1

(a) Cartoon representation of the crystal structure of NTL9_1-39 K12M (Protein Data Bank 2HBA). (Red, yellow, and green colors) The α-helix, the β-sheet, and the β-turn regions, respectively. The amide units with ¹³C=¹⁸O isotope labels are indicated on the structure. (b) Rotated view showing the G16 label as well as the V9 and G13 labels in the β-turn region. To see this figure in color, go online.

Figure 2

(Left) Network plot of the Markov state model for the native ensemble of NTL9_1-39. The states are color-coded by RMSD to the crystal structure. The node size encodes the degree, or number of connections to other states, of each state. (Dashed circle) Registry-shifted states (34). (Center) Structure overlay of the four highest populated states. The main differences are observed in the β-turn region. (Right) Nodes color-coded by the pseudo free energy of native states F = −ln(P), where P denotes the respective populations in the full MSM. To see this figure in color, go online.

Figure 3

Two-dimensional infrared spectra in the label region of the five different isotope labels of NTL9_1-39. (Dashed lines in the experimental spectrum) Positions of the diagonal slices. To see this figure in color, go online.

Figure 4

Diagonal slices of 2D IR spectra along the peak maxima in the isotope-label region (see Fig. 3). (Top) Experimental spectra. (Circles) Interpolated 2D IR data; (solid curves) dual Gaussian fits to the data as shown in Table 1. Gaussian center frequencies are indicated below each peak. All spectra are normalized to the maximum amplitude of the main amide I band. (Bottom) Simulated diagonal slices of the 2D IR spectra for the same labels. The G13 label (dashed curve) does not appear in the experimental spectrum, but it is included for comparison with V9 and V9G13 experimental curves. To see this figure in color, go online.

Figure 5

(a) Representative structures of the folded β-turn and disordered turn conformations represented in the MSM of NTL9_1-39 overlaid onto a cartoon structure of the Markov state with lowest RMSD to the crystal structure. (Spheres) Carbonyls corresponding to the V9 and G13 residues. (Yellow dashes) The two folded hydrogen bonds. (Orange double-headed arrow) The V9-M12 hydrogen bond used as the order parameter to distinguish between folded and disordered turn structures. (b) V9-, G13-, and V9G13-label spectra calculated for folded (solid) and disordered (dashed) structures, respectively. (c) Scatter plot of the V9-G13 coupling constant in wavenumbers as a function of r_VM distance for the 140 states in the MSM. (Circles are color-coded by the overall RMSD of each state to the crystal structure.) To see this figure in color, go online.

Figure 6

Hydrogen-bond analysis of the folded and disordered transitions. (a) Total number of hydrogen bonds accepted by V9(O) separated by protein-protein H-bonds n_PP (red) and protein-water H-bonds n_PW (blue). (b) Plot of backbone exposure calculated as the ratio of protein-water to total hydrogen bonds n_PW/(n_PP + n_PW). (Dashed lines) Linear fits to guide the eye. (c) MSM showing states color-coded by r_VM; (yellow) folded states; (red) disordered states. To see this figure in color, go online.

Figure 7

(a) Structures of the folded and solvent-exposed G24 configurations of the α-helix aligned to the lowest RMSD structure (black outline). (Red) The G24 carbonyls. (b) Simulated diagonal 2D IR spectra of the two conformations. The folded peak is more intense compared to solvent-exposed conformations. (c) MSM color-coded by G24(O) solvent exposure: n_PW/n_TOT. To see this figure in color, go online.