Computational and single-molecule force studies of a macro domain protein reveal a key molecular determinant for mechanical stability

Abstract

Resolving molecular determinants of mechanical stability of proteins is crucial in the rational design of advanced biomaterials for use in biomedical and nanotechnological applications. Here we present an interdisciplinary study combining bioinformatics screening, steered molecular dynamics simulations, protein engineering, and single-molecule force spectroscopy that explores the mechanical properties of a macro domain protein with mixed alpha + beta topology. The unique architecture is defined by a single seven-stranded beta-sheet in the core of the protein flanked by five alpha-helices. Unlike mechanically stable proteins studied thus far, the macro domain provides the distinct advantage of having the key load-bearing hydrogen bonds (H bonds) buried in the hydrophobic core protected from water attacks. This feature allows direct measurement of the force required to break apart the load-bearing H bonds under locally hydrophobic conditions. Steered molecular dynamics simulations predicted extremely high mechanical stability of the macro domain by using constant velocity and constant force methods. Single-molecule force spectroscopy experiments confirm the exceptional mechanical strength of the macro domain, measuring a rupture force as high as 570 pN. Furthermore, through selective deletion of shielding peptide segments, we examined the same key H bonds under hydrophilic environments in which the beta-strands are exposed to solvent and verify that the high mechanical stability of the macro domain results from excellent shielding of the load-bearing H bonds from competing water. Our study reveals that shielding water accessibility to the load-bearing strands is a critical molecular determinant for enhancing the mechanical stability of proteins.

Fig. 1.

Macro domain Af1521 topology. (A) A single 7-stranded β-sheet (yellow) is flanked by α-helices (purple). The disulfide and free cysteine residues are depicted in orange. The load-bearing strands are highlighted in blue and pulling atoms in red; (B) β-sheet displayed with strand order 1276354; and (C) top view of spiral β-strands.

Fig. 2.

CV-SMD unfolding trajectory of Af1521_11–177 at v = 0.1 Å ps^-1. (Inset) Force-extension profile of macro domain (purple) and I27 (orange). (A) Starting structure, x_11–177 = 28 Å; (B) snapshot of primary unfolding barrier involving strands 2 and 7, x_11—177 = 35 Å, F ≈ 2,600 pN; (C) macro domain unraveling without resistance until reaching the intermediate structure; (D) snapshot of secondary barrier involving strands 3 and 5 and the disulfide, x_11–177 = 213 Å, F ≈ 2,300 pN; and (E) the macro domain unravels to full extension (x_11–177 = 480 Å).

Fig. 3.

Trajectory analysis for Af1521_11–177 at constant force of 1,500 pN (CF-1500-1). (A) Representative snapshots illustrating water interaction with bond-breaking events between strands 2 and 7 at Δx = 4–7 Å (water shown in green); (B) load-bearing strands depicting amino acids involved in forming the seven parallel H bonds; and (C) distance-time plot of the load-bearing H bonds.

Fig. 4.

AFM force-extension data of Af1521_11–177. (A) Representative force curves at 1,000 nm s^-1 with WLC fit showing multiple unfolding pathways; (B) contour length distribution where ΔL_n = 45 nm, and ΔL₁ and ΔL₂ depict FΠI and IΠU transitions, respectively; (C) force histogram with Gaussian fit measuring 468 ± 44 pN at 1,000 nm s^-1; and (D) semilogarithmic plot of loading rate as a function of unfolding force (error bars represent standard deviation). A fit of the data to k(F) = k₀ exp(Fx_u/k_BT) (solid line) gives values of k₀ = 6.8 × 10^-3 s^-1 and x_u = 0.102 nm.

Fig. 5.

SMD analysis of -βαAf1521_11–177. (A) Structure of macro domain highlighting the shielding residues near the load-bearing strands in van der Waals representation; (B) CV-SMD force-extension profiles comparing unfolding forces of -βαAf1521_11–177 (green), Af1521_11–177 (purple), and I27 (orange); and (C) CF-1000 traces displaying the primary unfolding barrier at 5 Å (1°) and a secondary plateau at 11 Å (1°*). The four individual CF-1000 traces are labeled 1000-1, 1000-2, etc.

Fig. 6.

AFM force-extension data for -βαAf1521_11–177. (A) Representative SMFS traces at 1,000 nm s^-1 with WLC model fits (red lines) exhibiting evidence of intermediate formation (✓); (B) force histogram with Gaussian fit measuring an average force of 247 ± 76 pN; and (C) contour length distribution showing ΔL_c = 45 nm.