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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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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.
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pone-0073572-g003: 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.

Mentions: To provide an interpretation for those experiments, we used SMD simulations and we reproduced the extension process by keeping the C terminus fixed and moving the N terminus [26]. In order to perform these calculations in a reasonable time with such a large molecule, we used a structure-based coarse-grained model of Fha30. Therefore, it is difficult to compare the extension speed in the simulations with the experimental extension speed. The simulated F-D profiles obtained at different pulling speeds were similar, indicating a good convergence of the simulations (Fig. S1 in Supporting information). In spite of this limitation, the results from the two techniques were in reasonably good agreement. The simulated force-distance curves reproduced the main pattern of the experimental AFM profiles, exhibiting several small force peaks in the early part of the extension process, followed by several major force peaks of increasing forces (Fig. 3A). SMD simulations, thus, supported a multi-event unfolding reaction, in which regions of different stabilities unfolded successively (Fig. 3A). The different numbers of peaks between experiments and simulation may indicate that experimental unfolding proceeds by larger segments that likely encompass several consecutive in silico unfolding steps.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073572-g003: 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.
Mentions: To provide an interpretation for those experiments, we used SMD simulations and we reproduced the extension process by keeping the C terminus fixed and moving the N terminus [26]. In order to perform these calculations in a reasonable time with such a large molecule, we used a structure-based coarse-grained model of Fha30. Therefore, it is difficult to compare the extension speed in the simulations with the experimental extension speed. The simulated F-D profiles obtained at different pulling speeds were similar, indicating a good convergence of the simulations (Fig. S1 in Supporting information). In spite of this limitation, the results from the two techniques were in reasonably good agreement. The simulated force-distance curves reproduced the main pattern of the experimental AFM profiles, exhibiting several small force peaks in the early part of the extension process, followed by several major force peaks of increasing forces (Fig. 3A). SMD simulations, thus, supported a multi-event unfolding reaction, in which regions of different stabilities unfolded successively (Fig. 3A). The different numbers of peaks between experiments and simulation may indicate that experimental unfolding proceeds by larger segments that likely encompass several consecutive in silico unfolding steps.

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