Mechanical unfolding of an ankyrin repeat protein

Abstract

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins.

Figure 1

Constant-force simulations of the mechanical unfolding of gankyrin (1QYM) at 125 pN using FACTS implicit solvent. (A) Plot of extension versus time for a number of representative simulations. Unfolding occurs in a noncooperative manner, with multiple phases lasting for different periods of time. In some of the runs, the protein is trapped in a force-resistant intermediate state (red, blue, and violet traces) and in one run it does not reach the unfolded state within the duration of the simulation (black trace) (58 ns). Some other traces (in yellow and green) unfold very quickly without any long-lived intermediate state. (B) Left: Structure of the intermediate formed in the blue trajectory in panel A. The intermediate results from the unfolding of the most C-terminal repeat and part of the adjacent repeat. There is some disruption of the native packing in the structured regions, and nonnative contacts begin to form. Right: Structure of the long-lived intermediate formed in the black trajectory in panel A. The intermediate results from the unfolding of the three C-terminal ankyrin repeats and is stabilized by the formation of a nonnative parallel β-sheet formed between the long loops connecting two adjacent repeats (in yellow).

Figure 2

Representation of the distribution of extension lengths in the constant-force simulations. The upper panel shows the consensus ankyrin repeat NI3C, and the bottom panel shows gankyrin. Bars indicate the spacing corresponding to the length of one unfolded ankyrin repeat, and arrows indicate intermediates in which an integer number of domains is fully extended. Although they are more rare, conformations in which half a repeat is extended also exist. For gankyrin, the peaks are broad and their periodicity is not clearly related to the extension of individual domains.

Figure 3

Histograms of the extension length increments (ΔLc) between consecutive metastable states for gankyrin (left panel) and NI3C (right panel). Both proteins show two major increments, corresponding to 5–7.5 nm (half of an ankyrin repeat) and 10–12.5 nm (one ankyrin repeat). However, gankyrin shows other high-frequency increments ranging from ∼12.5 nm to ∼36 nm.

Figure 4

Histogram of the extension length increments (ΔLc) of the unfolding peaks of gankyrin in the polyprotein I27GKN measured by AFM. Forty-two force-extension curves, containing a total of 170 unfolding peaks of the gankyrin portion of the construct, were fitted to a WLC model of polymer elasticity and the ΔLc values were extracted. (A) Force-extension plot containing the unfolding of five I27 domains and gankyrin. The unfolding traces were fitted to a WLC model of polymer elasticity and the ΔLc values were extracted. The fittings for the I27 domains are represented by dotted lines. The fittings for gankyrin are represented by dashed lines. (B) A different trace containing the unfolding of five I27 domains and gankyrin. (C) Superimposition of traces A and B, showing a very accurate matching. (D) Histogram for the ΔLc values. The most probable events correspond to the unfolding of one or a half ankyrin repeat. However, extensions corresponding to the unfolding of two or more repeats are also highly represented.

Figure 5

Examples of unfolding and refolding force-extension relationships measured by AFM on the same gankyrin molecule in a cyclic measurement. (A) The first unfolding cycle (green) and refolding (blue) captured three clear ∼60–80 pN unfolding peaks at ∼5, 20, and 40 nm of the extension, and a ∼15 pN refolding peak (black star). (B) The molecule was lifted by 5 nm away from the substrate and stretched and relaxed again. The two unfolding force peaks (shown in red) overlap with the original unfolding force peaks (shown in green), and the relaxing trace (blue) captured one refolding force peak (black star). (C) The next stretching cycle (red) captured a single ∼200 pN unfolding force peak and the relaxing trace captured a small refolding event (black star). (D) A subsequent pulling cycle revealed ∼50 pN unfolding force peaks similar to those measured during the first unfolding cycle (in panel A). (E) Superimposition of force-extension curves of the gankyrin monomer and the I27GNK construct. Of note, gankyrin is shifted to the right to account for the extension of the folded polyprotein, showing a very accurate matching.