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

Alsteens D, Martinez N, Jamin M, Jacob-Dubuisson F - PLoS ONE (2013)

Bottom Line: In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified.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.

View Article: PubMed Central - PubMed

Affiliation: Université catholique de Louvain, Institute of Condensed Matter and Nanosciences, Louvain-la-Neuve, Belgium. david.alsteens@uclouvain.be

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.

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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.
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pone-0073572-g005: 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.

Mentions: Superposition of F-D curves of the two proteins was performed using a correlation analysis between the bivariate plots (Fig. 5A,B). To this end, we extracted the three major peaks of Fha30 (Fig. 5A), and the Fha30 plot was superimposed on the plot of Fha60 and translated progressively to determine the position for optimal superposition (Fig. 5B). Thus, for each relative position of the two plots (called hereafter translational shift), we normalized the corresponding matrix and multiplied the normalized frequency values of Fha30 with those corresponding to Fha60. We obtained a new matrix and we summarized the results as a column by summing up the results for each line. The same procedure was repeated after each translation step of the Fha30 plot relative to the Fha60 plot. The data are displayed as a 3D plot (Fig. 5C). The maxima of this plot reveal that the best alignment is for a translational shift of 150 nm. Since the part of the Fha30 plot used in the superposition procedure starts at an Lc value of 60 nm, the best alignment between the unfolding pathways of Fha30 and Fha60 occurs for a translational shift of 90 nm (Fig. 5D). Thus, using this method, we determined that the two major unfolding peaks of Fha30 correspond to peaks of Fha60 with Lc values of 160 nm and 190 nm (Fig. 5E). Those peaks correspond to substructures endowed with a high mechanical resistance in Fha30, and our analysis indicates that they unfold late in the unfolding pathway of Fha60 as well. The central part of the TPS domain thus appears to be a mechanically resistant portion of FHA. A second region of high mechanical resistance that unfolds in the last steps of the extension is found in Fha60. We also observed that initial force peaks of Fha60 at lower extensions are significantly higher than those of Fha30. We hypothesize that these differences originate from a stronger adsorption of the long molecule on the support, as FHA is known to adhere to abiotic surfaces [27].


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

Alsteens D, Martinez N, Jamin M, Jacob-Dubuisson F - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756990&req=5

pone-0073572-g005: 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.
Mentions: Superposition of F-D curves of the two proteins was performed using a correlation analysis between the bivariate plots (Fig. 5A,B). To this end, we extracted the three major peaks of Fha30 (Fig. 5A), and the Fha30 plot was superimposed on the plot of Fha60 and translated progressively to determine the position for optimal superposition (Fig. 5B). Thus, for each relative position of the two plots (called hereafter translational shift), we normalized the corresponding matrix and multiplied the normalized frequency values of Fha30 with those corresponding to Fha60. We obtained a new matrix and we summarized the results as a column by summing up the results for each line. The same procedure was repeated after each translation step of the Fha30 plot relative to the Fha60 plot. The data are displayed as a 3D plot (Fig. 5C). The maxima of this plot reveal that the best alignment is for a translational shift of 150 nm. Since the part of the Fha30 plot used in the superposition procedure starts at an Lc value of 60 nm, the best alignment between the unfolding pathways of Fha30 and Fha60 occurs for a translational shift of 90 nm (Fig. 5D). Thus, using this method, we determined that the two major unfolding peaks of Fha30 correspond to peaks of Fha60 with Lc values of 160 nm and 190 nm (Fig. 5E). Those peaks correspond to substructures endowed with a high mechanical resistance in Fha30, and our analysis indicates that they unfold late in the unfolding pathway of Fha60 as well. The central part of the TPS domain thus appears to be a mechanically resistant portion of FHA. A second region of high mechanical resistance that unfolds in the last steps of the extension is found in Fha60. We also observed that initial force peaks of Fha60 at lower extensions are significantly higher than those of Fha30. We hypothesize that these differences originate from a stronger adsorption of the long molecule on the support, as FHA is known to adhere to abiotic surfaces [27].

Bottom Line: In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified.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.

View Article: PubMed Central - PubMed

Affiliation: Université catholique de Louvain, Institute of Condensed Matter and Nanosciences, Louvain-la-Neuve, Belgium. david.alsteens@uclouvain.be

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.

Show MeSH
Related in: MedlinePlus