Residue-level interrogation of macromolecular crowding effects on protein stability

Abstract

Theory predicts that macromolecular crowding affects protein behavior, but experimental confirmation is scant. Herein, we report the first residue-level interrogation of the effects of macromolecular crowding on protein stability. We observe up to a 100-fold increase in the stability, as measured by the equilibrium constant for folding, for the globular protein chymotrypsin inhibitor 2 (CI2) in concentrations of the cosolute poly(vinylpyrrolidone) (PVP) that mimic the protein concentration in cells. We show that the increased stability is caused by the polymeric nature of PVP and that the degree of stabilization depends on both the location of the individual residue in the protein structure and the PVP concentration. Our data reinforce the assertion that macromolecular crowding stabilizes the protein by destabilizing its unfolded states.

Figure 1

PVP40 slows amide proton exchange. Exchange curves for the amide protons of Leu 32 (●) and Asp 52 (▼) in dilute solution (green) and 300 g/L PVP40 (cyan). Conditions: 50 mM acetate buffer in D2O, pH 5.4, 37 °C.

Figure 2

The I29A/I37H variant exchanges according to the EX2 mechanism. Linear regression yields slopes and R² values of 0.91 ± 0.5 and 0.92 in dilute solution (green) and 1.10 ± 0.08 and 0.86 in 300 g/L PVP40 (cyan), respectively. The error bars represent the standard errors for the averages from three trials collected at pH 5.4.

Figure 3

Crowding with 300 g/L PVP40 does not change exchange rate for loop residue 37, which is essentially unprotected, but adding 100 g/L NEP increases the rate approximately 4-fold. The exchange of residue 37 was measured in 50 mM acetate, pH 5.4 at 37 °C in 300 g/L PVP40 (cyan), in dilute solution (green), and in 100 g/L NEP (magenta) using the CLEANEX-PM experiment. The rate is 9 ± 2 s⁻¹ in dilute solution, 7 ± 1 s⁻¹ in PVP40, and 32 ± 3 s⁻¹ in NEP. The smooth curve is determined as described by Hwang et al.

Figure 4

Macromolecular crowding with PVP40 stabilizes the I29A/I37H variant of CI2 relative to dilute solution. Values of Δ $G_{\text{op}}^{\circ \prime }$ in 300 g/L PVP40 (cyan) and dilute solution (green) are plotted versus residue number. The height of each bar represents the average from three trials. The error bars represent the standard deviation. Conditions: 700–800 μM variant protein, 50 mM acetate buffer in D₂O, pH 5.4. The inset shows the backbone structure of PVP.

Figure 5

Ribbon structure of wild-type CI2 (PDB, 2CI2) colored by Δ $G_{\text{op}}^{\circ \prime }$ for the I29A/I37H variant in (A) dilute solution, (B) 300 g/L PVP40, and (C) 100 g/L NEP [blue: Δ $G_{\text{op}}^{\circ \prime }$ < 3.0 kcal/mol; green: < 3.0 kcal/mol Δ $G_{\text{op}}^{\circ \prime }$ < 5.0 kcal/mol; yellow: 5.0 kcal/mol < Δ $G_{\text{op}}^{\circ \prime }$ < 5.5 kcal/mol; cyan: 5.5 kcal/mol Δ $G_{\text{op}}^{\circ \prime }$ < 8.0 kcal/mol]. (D) Δ $G_{\text{op}}^{\circ \prime }$ was measured in 0, 50, 100, and 300 g/L PVP40, and δΔ $G_{\text{op}}^{\circ \prime }$ δ[PVP40] Values were superimposed onto the structure [blue: δΔ $G_{\text{op}}^{\circ \prime }$/δ[PVP40] = 0; yellow: 0 δΔ $G_{\text{op}}^{\circ \prime }$/δ[PVP40] < 2.5 × 10⁻³ (kcal/mol)/M; cyan: 2.5 × 10⁻³ (kcal/mol)/M < δΔ $G_{\text{op}}^{\circ \prime }$/δ[PVP40] < 1.0 × 10⁻² (kcal/mol)/M]. Residues for which there are no data are colored gray.