Correlation between chemical denaturation and the unfolding energetics of Acanthamoeba actophorin

Abstract

The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric and filamentous actin. Recent work on the protein actophorin from the amoeba Acanthamoeba castellani identified a series of site-directed mutations that increase the thermal stability of the protein by 22°C. Here, we expand this observation by showing that the mutant protein is also significantly stable to both equilibrium and kinetic chemical denaturation, and employ computer simulations to account for the increase in thermal or chemical stability through an accounting of atomic-level interactions. Specifically, the potential of mean force (PMF) can be obtained from steered molecular dynamics (SMD) simulations in which a protein is unfolded. However, SMD can be inefficient for large proteins as they require large solvent boxes, and computationally expensive as they require increasingly many SMD trajectories to converge the PMF. Adaptive steered molecular dynamics (ASMD) overcomes the second of these limitations by steering the particle in stages, which allows for convergence of the PMF using fewer trajectories compared with SMD. Use of the telescoping water scheme within ASMD partially overcomes the first of these limitations by reducing the number of waters at each stage to only those needed to solvate the structure within a given stage. In the PMFs obtained from ASMD, the work of unfolding Acto-2 was found to be higher than the Acto-WT by approximately 120 kCal/mol and reflects the increased stability seen in the chemical denaturation experiments. The evolution of the average number of hydrogen bonds and number of salt bridges during the pulling process provides a mechanistic view of the structural changes of the actophorin protein as it is unfolded, and how it is affected by the mutation in concert with the energetics reported through the PMF.

Figure 1

(A) Primary sequence comparison of Acto-WT and Acto-2. Sequence differences are highlighted. Identical amino acids are denoted by asterisks, conserved amino acids are denoted by colon, less conserved by period and nonconserved by blank. (B) Secondary structure sequence in Acto-2. $\beta$ Strands are indicated by cyan arrows. $\alpha$ Helices are indicated by purple rectangle. Individual $\alpha$ helices and $\beta$ strands are identified and referred to in text by the identifier shown in the parentheses above the purple rectangles and cyan arrows, respectively. (C) Three-dimensional superpositions of Acto-WT (PDB: 1AHQ; red) and Acto-2 (PDB: 7SOG; blue). To see this figure in color, go online.

Figure 2

Adaptive steered molecular dynamics with telescoping water box workflow. To see this figure in color, go online.

Figure 3

Viscosity reduction in F-actin network in presence of 10 $\mu$M Acto-WT (orange) and Acto-2 (blue). Actin was polymerized at 100 $\mu$M. F-Actin alone control (black). Plot represents average of three trials. To see this figure in color, go online.

Figure 4

(A) Fraction of unfolded of Acto-WT (orange) and Acto-2 (blue) as a function of GdHCl concentration. (B) The maximum emission wavelength for Acto-WT (orange) and Acto-2 (blue) versus GdHCl concentration. The reported values are the average of three independent experiments. To see this figure in color, go online.

Figure 5

(A) Fluorescence detected unfolding of Acto-2 by the rapid addition of 5 M GdHCl to a final concentration of 4.5 M. Data were fit to a single exponential function according to Eq. 5 in methods. (B) Fluorescence detected unfolding of Acto-WT by the rapid addition of 3 M GdHCl to a final concentration of 2.7 M. Data were fit to a single exponential function according to Eq. 5 in methods. Lower panels (C) and (D) show residuals for each fit. Each experimental curve is the average of 20 separate traces. To see this figure in color, go online.

Figure 6

(A) PMFs of Acto-2 (blue) and Acto-WT (orange). (B) The difference $\Delta \Delta U_{PMF}$ in the PMFs of Acto-2 minus Acto-WT. To see this figure in color, go online.

Figure 7

(A) Structure of Acto-WT and (B) structure of Acto-2 at $\Delta r_{ee}$ of 30, 80, 130, 180, and 200 Å. $\alpha$ Helices are colored purple, 3₁₀ helix blue, $\beta$ sheets cyan, coils black, and turns gray. To see this figure in color, go online.

Figure 8

Average number of fraction of native contacts for Acto-2 (blue) and Acto-WT (orange) as a function of pulling distance. To see this figure in color, go online.

Figure 9

Average number of (A) total, (B) 3₁₀, (C) $\alpha$, and (D) $\pi$ helix hydrogen bonds for Acto-2 (blue) and Acto-WT (orange) as a function of the pulling distance, $\Delta r_{ee}$.The error bars for the WT (red) and mutant (blue) represent the Jarzynski weighted cumulative error over the ensemble of 100 nonequilibrium trajectories determined at the end of each stage. To see this figure in color, go online.

Figure 10

Comparison of structures between Acto-WT (A and C) and Acto-2 (B and D) near site 21 (A and B) and site 22 (C and D). Mutated residues indicated in red. To see this figure in color, go online.

Figure 11

Number of salt bridges in Acto-WT and Acto-2 verses $\Delta r_{ee}$ for the trajectory closest to JE. The dark blue and dark orange lines represent the average number of salt bridges in each stage while the lighter blue and orange lines show the instantaneous fluctuations in the number of salt bridges during a stage. To see this figure in color, go online.