Sequential unfolding of beta helical protein by single-molecule atomic force microscopy

Abstract

The parallel βhelix is a common fold among extracellular proteins, however its mechanical properties remain unexplored. In Gram-negative bacteria, extracellular proteins of diverse functions of the large 'TpsA' family all fold into long βhelices. Here, single-molecule atomic force microscopy and steered molecular dynamics simulations were combined to investigate the mechanical properties of a prototypic TpsA protein, FHA, the major adhesin of Bordetella pertussis. Strong extension forces were required to fully unfold this highly repetitive protein, and unfolding occurred along a stepwise, hierarchical process. Our analyses showed that the extremities of the βhelix unfold early, while central regions of the helix are more resistant to mechanical unfolding. In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified. This nucleus harbors structural elements packed against the βhelix that might contribute to stabilizing the N-terminal region of FHA. Hierarchical unfolding of the βhelix in response to a mechanical stress may maintain β-helical portions that can serve as templates for regaining the native structure after stress. The mechanical properties uncovered here might apply to many proteins with β-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.

Figure 1. Schematic representation of FHA and model proteins used in this work.

(A) Model of FHA. The dotted vertical lines at the right side of the protein model indicate the approximate lengths of Fha60 and Fha30, which both have the same N-terminus as full-length FHA. The TPS domain is shown in blue, the R1 region is shown in red, and the B1, R2 and B2 domains are shown in green, magenta and cyan, respectively. The X-ray structure of Fha30 is known, while the R1 and R2 regions were built by using the models reported in , and the B1 and B2 regions were built by molecular modeling using the I-TASSER web server . (B) Conserved regions of Fha30. On the basis of multiple sequence alignments, four regions of different conservation rates were identified in the TPS domain. The most conserved subdomains C1 and C2 are shown in dark blue, and the less conserved subdomains LC1 and LC2 are shown in light blue. The first R1 coils are in red. (C) Structural organization of Fha30. The N-terminal cap and the six successive coils (Coil_A to Coil_F) of the TPS domain are shown in magenta, blue, cyan, green, yellow, orange and brown, respectively. The three R1 coils present in the crystal structure of Fha30 are shown in dark red, red and pink, and the three extra-helical elements are shown in grey. Elements II and III assemble together to form a four-stranded βsheet that packs against the β-helical core formed by coils_A to _F.

Figure 2. Unfolding of Fha30 by single-molecule AFM.

(A) Experimental set-up. (B) Representative force-distance curves obtained by stretching a single polypeptide, showing periodic features reflecting sequential unfolding peaks. Force peaks were well described by the worm-like-chain model (red line with Lc (nm)), using a persistence length of 0.4 nm. The curves shown are representative of a total of more than 200 adhesives curves obtained using 5 independent tips and 5 sample preparations. (C) Superposition of 25 typical force curves showing that the last three or four force peaks in particular are reproducibly observed. (D) Histograms of contour length Lc (n = 165) of the different peaks with Gaussian fit and statistics (mean ± SD). (E,F) Bivariate color-coded contour plots of Lc-ΔLc pairs for every individual unfolding event. Blue and red represent low and high frequencies of events, respectively.

Figure 3. Unfolding of Fha30 using simulated molecular dynamics.

(A) Comparison of a typical experimental force curve with a simulated F-D unfolding curve. Steered molecular dynamics simulation was performed using a coarse-grained model of the protein structure without N- and C-terminal tags (304 residues). The colored arrows indicate the positions along the simulation where contact maps were calculated (Fig. S2 in Supporting information). (B) Representation of the Fha30 structure showing the structural elements (colored according to the arrows in panel A) that unfolded in the successive force peaks. (C) Series of snapshots along the SMD unfolding trajectory. The structural elements are colored according to the events observed in the F-D curve and numbered in panel in A. The colored arrows indicate the regions of the protein that unfolded in each of the successive force peaks.

Figure 4. Unfolding of Fha60 by single-molecule AFM.

(A) Experimental set-up. (B) Representative force-distance curves obtained by stretching single polypeptides, showing periodic features reflecting sequential unfolding peaks. Force peaks were well described by the WLC model (red line with Lc (nm)), using a persistence length of 0.4 nm. The curves shown are representative of a total of more than 200 adhesives curves obtained using 5 independent tips and 5 sample preparations. (C) Superposition of 30 typical force curves showing that the last six force peaks in particular are reproducibly observed. (D) Histograms of contour length Lc (n = 171) of the different peaks with Gaussian fit and statistics (mean ± SD). (E,F) Bivariate color-coded contour plots of Lc-ΔLc pairs for every individual unfolding event. Blue and red represent low and high frequencies of events, respectively.

Figure 5. Superposition of the unfolding patterns of Fha30 and Fha60.

Comparison between the three major peaks of Fha30 [Lc = 60–140 nm] (A) and the full Fha60 (B) bivariate plots. (C) Normalized product between the two bivariate matrices reveals a maximum for a translational shift of 150 nm. (D) The best alignment between the two proteins occurs for a shift of 90 nm (150 nm minus the first 60 nm from the Fha30 bivariate plot). (E) Superposition of the force extension curves of the two proteins with the calculated best alignment.