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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.

No MeSH data available.


Single-molecule force microscopy and protein structure. (a) IFM31–33. The position of the cantilever tip was controlled using laser-radiation pressure. Feedback was applied such that the total forces exerted on the cantilever were equilibrated (see Supplementary Information, Fig. S1). (b) The difference between mechanical stretching using AFM and IFM. Before (broken blue lines) and after (solid black lines) stretching experiments. (top) In an AFM experiment, displacement (Δz) and velocity (Δvc) of the cantilever were not equal to those of the terminus of the protein molecule (Δd and Δv). (bottom) In an IFM experiment, the terminus of the protein molecule was stretched directly by pulling up the cantilever tip at a constant rate vc. (c) The SNase crystallographic structure (Protein Data Bank identifier (PDB ID): 1STN) lacked disordered regions (Ala1–Lys5 and Glu142–Gln149)40. The red, light-blue and green ribbons represent α-helices, β-structures and turns, respectively. (d) Scheme for SNase unfolding. Secondary-structure notations: S1–S5 (β-structure) and H1–H3 (α-helix). SNase is composed of two domains: the amino (N)-terminal β-barrel domain consisting of S1–S5 and H1, and the C-terminal α-domain consisting of H2 and H3. The Ca2+ binding site (dotted circle) consists of two turns between S1 and S2 and between S3 and H1.
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f1-5_25: Single-molecule force microscopy and protein structure. (a) IFM31–33. The position of the cantilever tip was controlled using laser-radiation pressure. Feedback was applied such that the total forces exerted on the cantilever were equilibrated (see Supplementary Information, Fig. S1). (b) The difference between mechanical stretching using AFM and IFM. Before (broken blue lines) and after (solid black lines) stretching experiments. (top) In an AFM experiment, displacement (Δz) and velocity (Δvc) of the cantilever were not equal to those of the terminus of the protein molecule (Δd and Δv). (bottom) In an IFM experiment, the terminus of the protein molecule was stretched directly by pulling up the cantilever tip at a constant rate vc. (c) The SNase crystallographic structure (Protein Data Bank identifier (PDB ID): 1STN) lacked disordered regions (Ala1–Lys5 and Glu142–Gln149)40. The red, light-blue and green ribbons represent α-helices, β-structures and turns, respectively. (d) Scheme for SNase unfolding. Secondary-structure notations: S1–S5 (β-structure) and H1–H3 (α-helix). SNase is composed of two domains: the amino (N)-terminal β-barrel domain consisting of S1–S5 and H1, and the C-terminal α-domain consisting of H2 and H3. The Ca2+ binding site (dotted circle) consists of two turns between S1 and S2 and between S3 and H1.

Mentions: The inter-/intramolecular-force microscopy (IFM) system31–33 (Fig. 1a) was based on an inverted microscope (IX70, Olympus) with an objective (PlanApo 60× oil, numerical aperture (NA) 1.4). A water-immersion objective (LUMPlanFl 40× W, NA 0.8, working distance (WD) 3.3 mm, Olympus) was installed above the specimen. Direct detection of cantilever displacement is essential for the feedback, instead of the detection of the angular change used in AFM; since the feedback position-control system needs detection of the absolute position of the cantilever tip, detection methods of angular change are unusable. The angle of the cantilever tip depends on not the absolute position but the relative position of the cantilever tip to the position of the cantilever root. We detected the displacement by forming a magnified image of the cantilever on a split-photodiode (magnification ~96×) by illumination with a red light-emitting diode (LED; L6112-01, Hamamatsu). The differential output of the photodiode was amplified and used as a feedback system (CF201, Sentech), which modulated the laser intensity (820 nm) for position control with a response frequency of 50 kHz. The maximum intensity of the laser was 30 mW, which exerted a force of ~100 pN on the cantilever. The displacement was calibrated by oscillating the cantilever base with a piezo actuator. As the direction of cantilever displacement was inclined at 45° from the optical axis, the detected displacement by the photosensor was proportional to cos (45°) times the displacement.


Stochastic emergence of multiple intermediates detected by single-molecule quasi-static mechanical unfolding of protein
Single-molecule force microscopy and protein structure. (a) IFM31–33. The position of the cantilever tip was controlled using laser-radiation pressure. Feedback was applied such that the total forces exerted on the cantilever were equilibrated (see Supplementary Information, Fig. S1). (b) The difference between mechanical stretching using AFM and IFM. Before (broken blue lines) and after (solid black lines) stretching experiments. (top) In an AFM experiment, displacement (Δz) and velocity (Δvc) of the cantilever were not equal to those of the terminus of the protein molecule (Δd and Δv). (bottom) In an IFM experiment, the terminus of the protein molecule was stretched directly by pulling up the cantilever tip at a constant rate vc. (c) The SNase crystallographic structure (Protein Data Bank identifier (PDB ID): 1STN) lacked disordered regions (Ala1–Lys5 and Glu142–Gln149)40. The red, light-blue and green ribbons represent α-helices, β-structures and turns, respectively. (d) Scheme for SNase unfolding. Secondary-structure notations: S1–S5 (β-structure) and H1–H3 (α-helix). SNase is composed of two domains: the amino (N)-terminal β-barrel domain consisting of S1–S5 and H1, and the C-terminal α-domain consisting of H2 and H3. The Ca2+ binding site (dotted circle) consists of two turns between S1 and S2 and between S3 and H1.
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Related In: Results  -  Collection

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f1-5_25: Single-molecule force microscopy and protein structure. (a) IFM31–33. The position of the cantilever tip was controlled using laser-radiation pressure. Feedback was applied such that the total forces exerted on the cantilever were equilibrated (see Supplementary Information, Fig. S1). (b) The difference between mechanical stretching using AFM and IFM. Before (broken blue lines) and after (solid black lines) stretching experiments. (top) In an AFM experiment, displacement (Δz) and velocity (Δvc) of the cantilever were not equal to those of the terminus of the protein molecule (Δd and Δv). (bottom) In an IFM experiment, the terminus of the protein molecule was stretched directly by pulling up the cantilever tip at a constant rate vc. (c) The SNase crystallographic structure (Protein Data Bank identifier (PDB ID): 1STN) lacked disordered regions (Ala1–Lys5 and Glu142–Gln149)40. The red, light-blue and green ribbons represent α-helices, β-structures and turns, respectively. (d) Scheme for SNase unfolding. Secondary-structure notations: S1–S5 (β-structure) and H1–H3 (α-helix). SNase is composed of two domains: the amino (N)-terminal β-barrel domain consisting of S1–S5 and H1, and the C-terminal α-domain consisting of H2 and H3. The Ca2+ binding site (dotted circle) consists of two turns between S1 and S2 and between S3 and H1.
Mentions: The inter-/intramolecular-force microscopy (IFM) system31–33 (Fig. 1a) was based on an inverted microscope (IX70, Olympus) with an objective (PlanApo 60× oil, numerical aperture (NA) 1.4). A water-immersion objective (LUMPlanFl 40× W, NA 0.8, working distance (WD) 3.3 mm, Olympus) was installed above the specimen. Direct detection of cantilever displacement is essential for the feedback, instead of the detection of the angular change used in AFM; since the feedback position-control system needs detection of the absolute position of the cantilever tip, detection methods of angular change are unusable. The angle of the cantilever tip depends on not the absolute position but the relative position of the cantilever tip to the position of the cantilever root. We detected the displacement by forming a magnified image of the cantilever on a split-photodiode (magnification ~96×) by illumination with a red light-emitting diode (LED; L6112-01, Hamamatsu). The differential output of the photodiode was amplified and used as a feedback system (CF201, Sentech), which modulated the laser intensity (820 nm) for position control with a response frequency of 50 kHz. The maximum intensity of the laser was 30 mW, which exerted a force of ~100 pN on the cantilever. The displacement was calibrated by oscillating the cantilever base with a piezo actuator. As the direction of cantilever displacement was inclined at 45° from the optical axis, the detected displacement by the photosensor was proportional to cos (45°) times the displacement.

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.