Quinary interactions with an unfolded state ensemble

Abstract

Anfinsen's thermodynamic hypothesis states that the native three-dimensional fold of a protein represents the structure with the lowest Gibbs free energy. Changes in the free energy of denaturation can arise from changes to the folded state, the unfolded state, or both. It has been recently recognized that quinary interactions, transient contacts that take place only in cells, can modulate protein stability through interactions involving the folded state. Here we show that the cellular environment can also remodel the unfolded state ensemble.

Keywords: NMR; amide proton exchange; folding; quinary structure; stability; thermodynamics; unfolded ensemble.

Figure 1

Stabilities in buffer. $\Delta G_{\text{op}}^{°\prime }$ values for the wild‐type (green), V21A (red), and V21I (blue) proteins. V21T (purple) was not stable enough to quantify, and the arrow indicates that the $\Delta G_{\text{op}}^{°\prime }$ is less than the least stable quantifiable residue (V29) in the wild‐type protein. The dashed lines indicate the mean $\Delta G_{\text{op}}^{°\prime }$ value, which equals $\Delta G_{\mathrm{U}}^{\mathrm{o}\prime }$, for each variant. $\Delta G_{\mathrm{U}}^{\mathrm{o}\prime }$ values are shown on the right with the standard deviation of the mean. $\Delta G_{\text{op}}^{°\prime }$ values were quantified as described.18, 35

Figure 2

Stabilities in cells. $\Delta G_{\text{op}}^{°\prime }$ values for wild‐type (green), V21A (red), and V21I (blue) in cells. The dashed lines indicate the mean $\Delta G_{\text{op}}^{°\prime }$ value, which equals $\Delta G_{\mathrm{U}}^{\mathrm{o}\prime }$. $\Delta G_{\mathrm{U}}^{\mathrm{o}\prime }$ values are shown on the right with the standard deviation of the mean. V21T (purple) was too destabilized to quantify. The arrows indicate that the $\Delta G_{\text{op}}^{°\prime }$ values are less than the least stable residue (V29) in the wild‐type protein. $\Delta G_{\text{op}}^{°\prime }$ values were quantified as described in the Supporting Information.

Figure 3

Mutations that increase GB1 stability in buffer have a small effect in cells. Changes in opening free energies, $\Delta \Delta G_{\text{op}}^{°\prime }$ ( $\Delta G_{\text{op},\text{var}}^{°\prime }-\Delta G_{\text{op},\text{wt}}^{°\prime }$) for GB1 V21A (top panel) and GB1 V21I (bottom panel) in buffer (blue) and in cells (green). Dashed lines indicate the $\Delta \Delta G_{\mathrm{U}}^{°\prime }$ value derived from the mean $\Delta \Delta G_{\text{op}}^{°\prime }$ values and their standard deviation.

Figure 4

Free energy diagram of the relative free energies for the folded (solid line) and unfolded states (dashed lines) in buffer (blue) and in cells (green). Folded states are arbitrarily assigned a common value.