Hydrogen bonding progressively strengthens upon transfer of the protein urea-denatured state to water and protecting osmolytes

Abstract

Using osmolyte cosolvents, we show that hydrogen-bonding contributions can be separated from hydrophobic interactions in the denatured state ensemble (DSE). Specifically, the effects of urea and the protecting osmolytes sarcosine and TMAO are reported on the thermally unfolded DSE of Nank4-7*, a truncated notch ankyrin protein. The high thermal energy of this state in the presence and absence of 6 M urea or 1 M sarcosine solution is sufficient to allow large changes in the hydrodynamic radius (R(h)) and secondary structure accretion without populating the native state. The CD change at 228 nm is proportional to the inverse of the volume of the DSE, giving a compact species equivalent to a premolten globule in 1 M sarcosine. The same general effects portraying hierarchical folding observed in the DSE at 55 degrees C are also often seen at room temperature. Analysis of Nank4-7* DSE structural energetics at room temperature as a function of solvent provides rationale for understanding the structural and dimensional effects in terms of how modulation of the solvent alters solvent quality for the peptide backbone. Results show that while the strength of hydrophobic interactions changes little on transferring the DSE from 6 M urea to water and then to 1 M TMAO, backbone-backbone (hydrogen-bonding) interactions are greatly enhanced due to progressively poorer solvent quality for the peptide backbone. Thus, increased intrachain hydrogen bonding guides secondary structure accretion and DSE contraction as solvent quality is decreased. This process is accompanied by increasing hydrophobic contacts as chain contraction gathers hydrophobes into proximity and the declining urea-backbone free energy gradient reaches urea concentrations that are energetically insufficient to keep hydrophobes apart in the DSE.

Figure 1

Temperature scans of Nank4−7* monitored by CD. Direct thermal scans and discrete points at 228 nm were taken to illustrate reproducibility. Thermal scans of Nank 4−7* are shown in the absence (recorded black line and open squares) and presence of sarcosine (recorded red lines) at 0.3 M (open circles), 0.6 M (open triangles), and 1 M concentrations (open inverted triangles). Also shown are thermal scans in urea (recorded blue lines) at 2 M (gray circles), 4 M (gray triangles), and 6 M (gray inverted triangles). All solutions contained 10 mM sodium phosphate buffer, pH 7.0, and 200 mM NaCl. Three conditions of interest at 55 °C, as explained in the text, are shown: (A) the extrapolated value for the native state baseline, (B) the thermally denatured state in the presence of 1 M sarcosine, and (C) the thermally denatured state in the presence of 6 M urea.

Figure 2

Relationship between hydrodynamic volume and CD. The hydrodynamic volume (degree of compactness) of Nank4−7* species thermally denatured at 55 °C (R_h^⊗)³ relative to the volume of thermally denatured Nank4−7* species in 0, 2, and 4 M urea (R_h)³. The ratio of these two parameters is plotted as a function of the corresponding θ₂₂₈ for the species of interest relative to θ₂₂₈^⊗ (the ellipticity of thermally denatured Nank4−7* in 0 M urea). The linear fit is defined by eq 1 in the text. From left to right, symbols are for 4, 2, and 0 M urea for thermally denatured (55 °C) Nank4−7* with native Nank4−7* (θ₂₂₈ and R_h determined at 15 °C) at the top right of the plot. The arrows indicate values of R_h extracted from the graph using the respective θ₂₂₈ values recorded for 0.3, 0.6, and 1.0 M sarcosine.

Figure 3

Full range of dimensional changes in Nank4−7* relative to literature values. (A) R_h and/or R_g as a function of the number of amino acids (N) in the protein. The solid line is a fit through all of the data with the 95% confidence limits (heavy dashed lines). The dot-dashed line is a fit through the R_g data from Kohn et al. (36) and the light dotted line a fit through the R_h data from Uversky (37). The open square in the plot represents the region expanded for panel B. (B) R_h values for Nank4−7* are reported in the context of a plot of R_h(37) and R_g(36) values as a function of protein chain length. R_h values reported for Nank4−7* species in 0.3, 0.6, and 1 M sarcosine solutions were calculated from their measured θ₂₂₈ values using eq 1. Key: filled circle, 4 M urea (33.4 Å); filled triangle, 2 M urea (31.8 Å); open triangle, thermally denatured state (29.5 Å); filled star, 0.3 M sarcosine (28.6 Å); cross, 0.6 M sarcosine (28.0 Å); filled diamond, 1 M sarcosine (26.6 Å); open star, native Nank4−7*.

Figure 4

Molar ellipticity for the Nank 4−7* denatured state at 61 °C in 10 mM sodium phosphate, pH 7.0, and 200 mM NaCl as a function of cosolvent concentration. Molar ellipticity at 228 nm is given as a function of urea and several protecting osmolyte concentrations including urea (open squares), sarcosine (circles), and TMAO (triangles). Nank 4−7* in the absence of the osmolytes is represented as a closed square.

Figure 5

Side chain and backbone contributions to ΔG_tr,D of Nank4−7* DSE from water to 1 M urea (A), sarcosine (B), and TMAO (C) solution. Side chain and backbone transfer free energy contributions (Δg_tr) divided by the corresponding surface areas exposed in the DSE are plotted as a function of those solvent-exposed surface areas. Backbone contributions are represented by the color dark green, whereas basic, acidic, polar, and nonpolar side chains are in blue, red, green, and yellow, respectively, and standard letters identify the side chains. The area under each bar is proportional to the transfer free energy contribution of that AA side chain to ΔG_tr,D. The denatured state model used is a self-avoiding random coil with side chain and backbone surface areas evaluated as described previously (3). Numerical values for side chains and backbone contributions are given in Table 2.

Figure 6

Nank4−7* DSE transfer free energy from 6 M urea (DSE_6Murea) to water (DSE_w). The left side shows the net transfer free energy change. The right side represents group contributions to the net free energy from transfer of the backbone to water (DSE_bb,w), along with transfer of those side chains destined for burial on protein folding (DSE_sc,B,w) and side chains that are solvent exposed in the native state (DSE_sc,N,w).