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Stochastic emergence of multiple intermediates detected by single-molecule quasi-static mechanical unfolding of protein

View Article: PubMed Central - PubMed

ABSTRACT

Experimental probing of a protein-folding energy landscape can be challenging, and energy landscapes comprising multiple intermediates have not yet been defined. Here, we quasi-statically unfolded single molecules of staphylococcal nuclease by constant-rate mechanical stretching with a feedback positioning system. Multiple discrete transition states were detected as force peaks, and only some of the multiple transition states emerged stochastically in each trial. This finding was confirmed by molecular dynamics simulations, and agreed with another result of the simulations which showed that individual trajectories took highly heterogeneous pathways. The presence of Ca2+ did not change the location of the transition states, but changed the frequency of the emergence. Transition states emerged more frequently in stabilized domains. The simulations also confirmed this feature, and showed that the stabilized domains had rugged energy surfaces. The mean energy required per residue to disrupt secondary structures was a few times the thermal energy (1–3 kBT), which agreed with the stochastic feature. Thus, single-molecule quasi-static measurement has achieved notable success in detecting stochastic features of a huge number of possible conformations of a protein.

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Related in: MedlinePlus

Ca2+ effect clarified by cluster analysis. (a–c) The mean force-extension curves of clusters in three groups. All 85 experimental force-extension curves (data) in both the absence and presence of Ca2+ were partitioned into 10 clusters by cluster analysis. The data in each cluster were averaged, and 10 mean force-extension curves of the clusters (shown in a–c) were obtained. The mean curves were used for further cluster analysis, and the 10 clusters were partitioned into 3 groups. Arrows indicate force peaks. (d–f) Number of data comprising each cluster in the group 1 (d), 2 (e), and 3 (f), respectively. Data were counted separately in the absence (left) and presence (right) of Ca2+. All 3 clusters in group 1 (black, red, blue) showed data 1.5 times or higher in the absence of Ca2+ than in the presence of Ca2+. In contrast, all 3 clusters in group 2 (black, red, blue) showed data 1.5 times or higher in the presence of Ca2+ than in the absence of Ca2+.
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f5-5_25: Ca2+ effect clarified by cluster analysis. (a–c) The mean force-extension curves of clusters in three groups. All 85 experimental force-extension curves (data) in both the absence and presence of Ca2+ were partitioned into 10 clusters by cluster analysis. The data in each cluster were averaged, and 10 mean force-extension curves of the clusters (shown in a–c) were obtained. The mean curves were used for further cluster analysis, and the 10 clusters were partitioned into 3 groups. Arrows indicate force peaks. (d–f) Number of data comprising each cluster in the group 1 (d), 2 (e), and 3 (f), respectively. Data were counted separately in the absence (left) and presence (right) of Ca2+. All 3 clusters in group 1 (black, red, blue) showed data 1.5 times or higher in the absence of Ca2+ than in the presence of Ca2+. In contrast, all 3 clusters in group 2 (black, red, blue) showed data 1.5 times or higher in the presence of Ca2+ than in the absence of Ca2+.

Mentions: To clarify the Ca2+ effect, we performed cluster analysis of all 85 experimental force-extension curves in both the absence and presence of Ca2+. The 85 force-extension curves were partitioned into 10 clusters. The mean force-extension curves of the 10 clusters were further divided into 3 groups (Fig. 5). The mean force-extension curves of the clusters in the groups 1 and 2 showed distinct force peaks, while those in group 3 did not. The force-peak amplitude of the mean force-extension curves in groups 1 and 2 was 5 times larger than that of the noise amplitude (Fig. 5a and b; see Methods section).


Stochastic emergence of multiple intermediates detected by single-molecule quasi-static mechanical unfolding of protein
Ca2+ effect clarified by cluster analysis. (a–c) The mean force-extension curves of clusters in three groups. All 85 experimental force-extension curves (data) in both the absence and presence of Ca2+ were partitioned into 10 clusters by cluster analysis. The data in each cluster were averaged, and 10 mean force-extension curves of the clusters (shown in a–c) were obtained. The mean curves were used for further cluster analysis, and the 10 clusters were partitioned into 3 groups. Arrows indicate force peaks. (d–f) Number of data comprising each cluster in the group 1 (d), 2 (e), and 3 (f), respectively. Data were counted separately in the absence (left) and presence (right) of Ca2+. All 3 clusters in group 1 (black, red, blue) showed data 1.5 times or higher in the absence of Ca2+ than in the presence of Ca2+. In contrast, all 3 clusters in group 2 (black, red, blue) showed data 1.5 times or higher in the presence of Ca2+ than in the absence of Ca2+.
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Related In: Results  -  Collection

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f5-5_25: Ca2+ effect clarified by cluster analysis. (a–c) The mean force-extension curves of clusters in three groups. All 85 experimental force-extension curves (data) in both the absence and presence of Ca2+ were partitioned into 10 clusters by cluster analysis. The data in each cluster were averaged, and 10 mean force-extension curves of the clusters (shown in a–c) were obtained. The mean curves were used for further cluster analysis, and the 10 clusters were partitioned into 3 groups. Arrows indicate force peaks. (d–f) Number of data comprising each cluster in the group 1 (d), 2 (e), and 3 (f), respectively. Data were counted separately in the absence (left) and presence (right) of Ca2+. All 3 clusters in group 1 (black, red, blue) showed data 1.5 times or higher in the absence of Ca2+ than in the presence of Ca2+. In contrast, all 3 clusters in group 2 (black, red, blue) showed data 1.5 times or higher in the presence of Ca2+ than in the absence of Ca2+.
Mentions: To clarify the Ca2+ effect, we performed cluster analysis of all 85 experimental force-extension curves in both the absence and presence of Ca2+. The 85 force-extension curves were partitioned into 10 clusters. The mean force-extension curves of the 10 clusters were further divided into 3 groups (Fig. 5). The mean force-extension curves of the clusters in the groups 1 and 2 showed distinct force peaks, while those in group 3 did not. The force-peak amplitude of the mean force-extension curves in groups 1 and 2 was 5 times larger than that of the noise amplitude (Fig. 5a and b; see Methods section).

View Article: PubMed Central - PubMed

ABSTRACT

Experimental probing of a protein-folding energy landscape can be challenging, and energy landscapes comprising multiple intermediates have not yet been defined. Here, we quasi-statically unfolded single molecules of staphylococcal nuclease by constant-rate mechanical stretching with a feedback positioning system. Multiple discrete transition states were detected as force peaks, and only some of the multiple transition states emerged stochastically in each trial. This finding was confirmed by molecular dynamics simulations, and agreed with another result of the simulations which showed that individual trajectories took highly heterogeneous pathways. The presence of Ca2+ did not change the location of the transition states, but changed the frequency of the emergence. Transition states emerged more frequently in stabilized domains. The simulations also confirmed this feature, and showed that the stabilized domains had rugged energy surfaces. The mean energy required per residue to disrupt secondary structures was a few times the thermal energy (1–3 kBT), which agreed with the stochastic feature. Thus, single-molecule quasi-static measurement has achieved notable success in detecting stochastic features of a huge number of possible conformations of a protein.

No MeSH data available.


Related in: MedlinePlus