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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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Shift in force-peak distribution by Ca2+ binding. (a–d) Distribution of force-peak locations in mean force-extension curves by cluster analysis (Fig. 5a–c) derived experimentally (a and c) and by MD simulation (b and d) in the absence (a and b) and presence (c and d) of Ca2+. The number of force peaks was weighted by the number of data comprising the clusters. Gray vertical lines represent peak locations in the distribution of force peaks without cluster analysis shown in Fig. 3a and b. (e and f) The ratio of the number of force peaks in the former and latter half of extension. The boundary distance was 23 nm for experimental data (see text on Fig. 5 results) and 25 nm for MD data (23+2 nm). Magenta, cyan and orange bars indicate that the force peak of interest emerged during unfolding of the α-domain, β-barrel domain, and other events, respectively in MD simulations.
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f6-5_25: Shift in force-peak distribution by Ca2+ binding. (a–d) Distribution of force-peak locations in mean force-extension curves by cluster analysis (Fig. 5a–c) derived experimentally (a and c) and by MD simulation (b and d) in the absence (a and b) and presence (c and d) of Ca2+. The number of force peaks was weighted by the number of data comprising the clusters. Gray vertical lines represent peak locations in the distribution of force peaks without cluster analysis shown in Fig. 3a and b. (e and f) The ratio of the number of force peaks in the former and latter half of extension. The boundary distance was 23 nm for experimental data (see text on Fig. 5 results) and 25 nm for MD data (23+2 nm). Magenta, cyan and orange bars indicate that the force peak of interest emerged during unfolding of the α-domain, β-barrel domain, and other events, respectively in MD simulations.

Mentions: The distribution of experimental force-peak locations of the mean force-extension curves by cluster analysis (Fig. 6a and c) showed several peaks in the same locations as those without cluster analysis (Fig. 3a and b). Cluster analysis was also applied to MD force-extension curves. The histogram of MD force-peak locations by cluster analysis (Fig. 6b and d) again showed a distribution similar to that without cluster analysis (Fig. 3c and d) and to the experimental histogram (Fig. 6a and c). Since force peaks correspond to transition states as mentioned above, this result indicates that the detection of multiple transition states in unfolding was confirmed by MD simulation and cluster analysis.


Stochastic emergence of multiple intermediates detected by single-molecule quasi-static mechanical unfolding of protein
Shift in force-peak distribution by Ca2+ binding. (a–d) Distribution of force-peak locations in mean force-extension curves by cluster analysis (Fig. 5a–c) derived experimentally (a and c) and by MD simulation (b and d) in the absence (a and b) and presence (c and d) of Ca2+. The number of force peaks was weighted by the number of data comprising the clusters. Gray vertical lines represent peak locations in the distribution of force peaks without cluster analysis shown in Fig. 3a and b. (e and f) The ratio of the number of force peaks in the former and latter half of extension. The boundary distance was 23 nm for experimental data (see text on Fig. 5 results) and 25 nm for MD data (23+2 nm). Magenta, cyan and orange bars indicate that the force peak of interest emerged during unfolding of the α-domain, β-barrel domain, and other events, respectively in MD simulations.
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Related In: Results  -  Collection

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f6-5_25: Shift in force-peak distribution by Ca2+ binding. (a–d) Distribution of force-peak locations in mean force-extension curves by cluster analysis (Fig. 5a–c) derived experimentally (a and c) and by MD simulation (b and d) in the absence (a and b) and presence (c and d) of Ca2+. The number of force peaks was weighted by the number of data comprising the clusters. Gray vertical lines represent peak locations in the distribution of force peaks without cluster analysis shown in Fig. 3a and b. (e and f) The ratio of the number of force peaks in the former and latter half of extension. The boundary distance was 23 nm for experimental data (see text on Fig. 5 results) and 25 nm for MD data (23+2 nm). Magenta, cyan and orange bars indicate that the force peak of interest emerged during unfolding of the α-domain, β-barrel domain, and other events, respectively in MD simulations.
Mentions: The distribution of experimental force-peak locations of the mean force-extension curves by cluster analysis (Fig. 6a and c) showed several peaks in the same locations as those without cluster analysis (Fig. 3a and b). Cluster analysis was also applied to MD force-extension curves. The histogram of MD force-peak locations by cluster analysis (Fig. 6b and d) again showed a distribution similar to that without cluster analysis (Fig. 3c and d) and to the experimental histogram (Fig. 6a and c). Since force peaks correspond to transition states as mentioned above, this result indicates that the detection of multiple transition states in unfolding was confirmed by MD simulation and cluster analysis.

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