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

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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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Intermediate structures and structural origins of force peaks obtained by MD simulations. Representative structures of intermediates from MD simulations in the absence (a) and presence (b) of Ca2+. Structures of interest before and after transition are shown as yellow solid and white broken circles, respectively. Histograms (inset) show distributions of force-peak locations in the force-extension relationships (as in Fig. 3c and d). Force peaks corresponded to the breaks of secondary structures or hydrophobic interactions.
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f4-5_25: Intermediate structures and structural origins of force peaks obtained by MD simulations. Representative structures of intermediates from MD simulations in the absence (a) and presence (b) of Ca2+. Structures of interest before and after transition are shown as yellow solid and white broken circles, respectively. Histograms (inset) show distributions of force-peak locations in the force-extension relationships (as in Fig. 3c and d). Force peaks corresponded to the breaks of secondary structures or hydrophobic interactions.

Mentions: To examine transition states and unfolding pathways, we carried out MD simulations of SNase stretching in the absence and presence of Ca2+ (50 simulations for each condition). MD force-extension showed similar profiles to those obtained experimentally (see Supplementary Information, Fig. S3). Force peaks corresponded to breaks in the secondary structure or hydrophobic interaction (Fig. 4). The histogram of force-peak locations from all simulations also showed a distribution similar to the experimental histogram (Fig. 3c and d). It was composed of five peaks, which largely agreed with the experimental results, except for the absence of a peak corresponding to the experimental 7-nm peak. This peak might have been brought about by stretching the terminal region (Ala1–Lys5 and Glu142–Gln149 in the absence of Ca2+; Ala1–Lys6 and Glu142–Gln149 in the presence of Ca2+), which is not included in the simulation, and might correspond to the finding that the carboxy (C)-terminal end region (Trp140–Gln149) is important for structural stability41. The best agreement was obtained when the MD axis of the end-to-end distance was shifted to the left by 2.0 nm (broken line in Fig. 3c and d). This might have been caused by anchoring the configuration of the protein termini on experimental surfaces. The agreements between experiments and simulations reflected the direct control of the extension and constant-rate stretching.


Stochastic emergence of multiple intermediates detected by single-molecule quasi-static mechanical unfolding of protein
Intermediate structures and structural origins of force peaks obtained by MD simulations. Representative structures of intermediates from MD simulations in the absence (a) and presence (b) of Ca2+. Structures of interest before and after transition are shown as yellow solid and white broken circles, respectively. Histograms (inset) show distributions of force-peak locations in the force-extension relationships (as in Fig. 3c and d). Force peaks corresponded to the breaks of secondary structures or hydrophobic interactions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036639&req=5

f4-5_25: Intermediate structures and structural origins of force peaks obtained by MD simulations. Representative structures of intermediates from MD simulations in the absence (a) and presence (b) of Ca2+. Structures of interest before and after transition are shown as yellow solid and white broken circles, respectively. Histograms (inset) show distributions of force-peak locations in the force-extension relationships (as in Fig. 3c and d). Force peaks corresponded to the breaks of secondary structures or hydrophobic interactions.
Mentions: To examine transition states and unfolding pathways, we carried out MD simulations of SNase stretching in the absence and presence of Ca2+ (50 simulations for each condition). MD force-extension showed similar profiles to those obtained experimentally (see Supplementary Information, Fig. S3). Force peaks corresponded to breaks in the secondary structure or hydrophobic interaction (Fig. 4). The histogram of force-peak locations from all simulations also showed a distribution similar to the experimental histogram (Fig. 3c and d). It was composed of five peaks, which largely agreed with the experimental results, except for the absence of a peak corresponding to the experimental 7-nm peak. This peak might have been brought about by stretching the terminal region (Ala1–Lys5 and Glu142–Gln149 in the absence of Ca2+; Ala1–Lys6 and Glu142–Gln149 in the presence of Ca2+), which is not included in the simulation, and might correspond to the finding that the carboxy (C)-terminal end region (Trp140–Gln149) is important for structural stability41. The best agreement was obtained when the MD axis of the end-to-end distance was shifted to the left by 2.0 nm (broken line in Fig. 3c and d). This might have been caused by anchoring the configuration of the protein termini on experimental surfaces. The agreements between experiments and simulations reflected the direct control of the extension and constant-rate stretching.

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