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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 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.
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pone-0073572-g002: 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.

Mentions: Fha30 was modified with an N-terminal 6-His tag and two Cys residues in tandem at the C terminus, and it was co-produced with its transporter FhaC in Escherichia coli and purified from culture supernatants. It was grafted to a gold surface through C-terminal Cys residues and picked up and stretched through the N-terminal 6-His tag using an AFM tip derivatized with NTA-Ni2+ (Fig. 2A). Recording force-distance (F-D) curves revealed that a small fraction of all recorded curves exhibited an adhesion event (∼2% from a total of more than 10000 curves). A substantial fraction of the adhesives curves (>40%) exhibited a saw-tooth pattern with successive force peaks, suggesting that the polypeptide chain experienced a multi-step unfolding process as a result of force application (Fig. 2B). In those curves, the unfolding process is characterized by an initial series of small force peaks situated in the first 50 nm. These initial forces peaks differ considerably from one curve to another (Fig. 2B), indicating that the first steps of unfolding may be influenced by non-specific interactions of the protein with the surface. The first region of the F-D curve is followed by several peaks of increasing forces above 100 nm. The gradual, non-linear force increases of the extension steps could be fitted with the worm-like chain (WLC) model using only one free parameter: the contour length (Lc) of the stretched portion of the molecule (Fig. 2B). These fits were used to determine the increases in protein contour length (ΔLc), which reflect the lengths of the protein segments that unfold in each consecutive event. The last peak reflects the extension of the polypeptide to a completely stretched state, where rupture of the bound between the His tag and the Ni2+-NTA group of the AFM tip occurs (Fig. 2C). The overall contour length (Lc) of 127 nm of Fha30 determined from this last peak is compatible with full extension of the protein assuming that each amino acid contributes 0.4 nm to the contour length of the fully extended polypeptide chain (the recombinant protein is 325 residues long, thus the expected length for the fully extended polypeptide is 0.4×325 = 130 nm). Superposition of independent force-extension curves (see Materials and Methods for details) showed that the three major unfolding peaks were almost always present, supporting a sequential process along which progressive forces hierarchically unfolded increasingly stable regions of the protein (Fig. 2C,D). This behavior is different from that generally observed for the individual domains of multi-domain proteins, such as titin, that unfold in a more concerted manner, in one or two force peaks [7]. Unfolding peaks of Fha30 occurred at applied forces of ≈125 to 250 pN, consistent with the unfolding of domains with βstructures [7] and much higher than for a-helical proteins [8]. Lc values for all the events were summarized in histograms, with Gaussian fits (mean ± SD, n = 165; Fig. 2D) revealing narrow distributions for the three major peaks of unfolding centered around 43±7 nm, 71±3 nm and 97±2 nm, and for the rupture peak at 127±3 nm. Since some force extension curves showed sawtooth patterns with some missing peaks, bivariate diagrams were used to link Lc values with the ΔLc values corresponding to the distances between each peak and the previous one (Fig. 2E,F). Maxima in the frequency of occurrence of the Lc/ΔLc pairs appear in red/yellow. Bivariate plots revealed that the Fha30 unfolding pathway presents two major unfolding peaks at Lc/?Lc values of 71 nm/32 nm and 97 nm/35 nm as well as a rupture peak at 127 nm/30 nm. The rupture peak was analyzed to determine the ?Lc of the last unfolding peak. Our analysis thus reveals that sequential unfolding, one segment after the other, is the most frequent unfolding pathway for Fha30.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756990&req=5

pone-0073572-g002: 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.
Mentions: Fha30 was modified with an N-terminal 6-His tag and two Cys residues in tandem at the C terminus, and it was co-produced with its transporter FhaC in Escherichia coli and purified from culture supernatants. It was grafted to a gold surface through C-terminal Cys residues and picked up and stretched through the N-terminal 6-His tag using an AFM tip derivatized with NTA-Ni2+ (Fig. 2A). Recording force-distance (F-D) curves revealed that a small fraction of all recorded curves exhibited an adhesion event (∼2% from a total of more than 10000 curves). A substantial fraction of the adhesives curves (>40%) exhibited a saw-tooth pattern with successive force peaks, suggesting that the polypeptide chain experienced a multi-step unfolding process as a result of force application (Fig. 2B). In those curves, the unfolding process is characterized by an initial series of small force peaks situated in the first 50 nm. These initial forces peaks differ considerably from one curve to another (Fig. 2B), indicating that the first steps of unfolding may be influenced by non-specific interactions of the protein with the surface. The first region of the F-D curve is followed by several peaks of increasing forces above 100 nm. The gradual, non-linear force increases of the extension steps could be fitted with the worm-like chain (WLC) model using only one free parameter: the contour length (Lc) of the stretched portion of the molecule (Fig. 2B). These fits were used to determine the increases in protein contour length (ΔLc), which reflect the lengths of the protein segments that unfold in each consecutive event. The last peak reflects the extension of the polypeptide to a completely stretched state, where rupture of the bound between the His tag and the Ni2+-NTA group of the AFM tip occurs (Fig. 2C). The overall contour length (Lc) of 127 nm of Fha30 determined from this last peak is compatible with full extension of the protein assuming that each amino acid contributes 0.4 nm to the contour length of the fully extended polypeptide chain (the recombinant protein is 325 residues long, thus the expected length for the fully extended polypeptide is 0.4×325 = 130 nm). Superposition of independent force-extension curves (see Materials and Methods for details) showed that the three major unfolding peaks were almost always present, supporting a sequential process along which progressive forces hierarchically unfolded increasingly stable regions of the protein (Fig. 2C,D). This behavior is different from that generally observed for the individual domains of multi-domain proteins, such as titin, that unfold in a more concerted manner, in one or two force peaks [7]. Unfolding peaks of Fha30 occurred at applied forces of ≈125 to 250 pN, consistent with the unfolding of domains with βstructures [7] and much higher than for a-helical proteins [8]. Lc values for all the events were summarized in histograms, with Gaussian fits (mean ± SD, n = 165; Fig. 2D) revealing narrow distributions for the three major peaks of unfolding centered around 43±7 nm, 71±3 nm and 97±2 nm, and for the rupture peak at 127±3 nm. Since some force extension curves showed sawtooth patterns with some missing peaks, bivariate diagrams were used to link Lc values with the ΔLc values corresponding to the distances between each peak and the previous one (Fig. 2E,F). Maxima in the frequency of occurrence of the Lc/ΔLc pairs appear in red/yellow. Bivariate plots revealed that the Fha30 unfolding pathway presents two major unfolding peaks at Lc/?Lc values of 71 nm/32 nm and 97 nm/35 nm as well as a rupture peak at 127 nm/30 nm. The rupture peak was analyzed to determine the ?Lc of the last unfolding peak. Our analysis thus reveals that sequential unfolding, one segment after the other, is the most frequent unfolding pathway for Fha30.

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