Data from: Force-induced ankle opening reveals mechanical stabilization of the ankle of human β-cardiac myosin
Data files
Jun 03, 2026 version files 53.03 MB
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Manuscript_Figures.zip
29.19 MB
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README.md
15.17 KB
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SI_Figures.zip
23.83 MB
Abstract
Human β-cardiac myosin drives contraction in the heart. Extensive biophysical and single-molecule studies have quantified myosin’s chemo-mechanical cycle, which generates ~5 nm of displacement and 5–7 pN of force. Myosin’s 9-nm-long, α-helical lever arm is rigidified by bound essential and regulatory light chains (ELC and RLC). Numerous pathogenic mutations and sequence-conservation patterns within the lever arm where the RLC binds (LARLC) belie the overly simplified view that the lever arm acts solely as a rigid rod that transduces ATP hydrolysis into motion. Structural studies have shown that myosin adopts an interacting-heads motif (IHM), which inhibits motor activity and mechanically strains the RLC complex, consisting of the RLC bound to the LARLC. Alteration in the configuration of the RLC complex’s “ankle”—a sharp kink in the lever arm—is hypothesized to modulate the propensity of myosin to enter the IHM. To investigate the complex’s mechanical stability, we developed a single-molecule atomic-force microscopy assay with three different pulling geometries: pulling across the LARLC, the RLC, and the RLC complex. When pulling across the LARLC by applying force to its N and C termini, the mechanical dissociation of the RLC was resolved along with two intermediates. Coarse-grained Brownian dynamics detailed these molecular configurations as the opening of myosin’s ankle and the preferential dissociation of one of the RLC’s two EF-hand domains. Moreover, the linker between the EF-hand domains forms an interface with an RLC N-terminal loop. This interface stabilized the native acute ankle angle against opening. Pulling across the RLC and the RLC complex revealed different unfolding pathways, each with one intermediate. Looking forward, these assays can probe for the effects of pathogenic mutations and phosphorylation on the nanomechanics of the RLC complex.
Dataset DOI: 10.5061/dryad.mpg4f4rg9
Description of the data and file structure
Files and variables
File: Manuscript_Figures.zip
Description: Contains .csv files for figures in the manuscript
File: SI_Figures.zip
Description: Contains .csv files for figures in the SI.
Units:
- N (newtons) or pN (piconewtons) for force
- m (meters) or nm (nanometers) for distance
- s (seconds) or us (microseconds) for time
- degrees for angles
Note: Data for zoomed-in insets is taken from their respective zoomed-out figures.
-Data is provided in .csv format.
-Each figure has its own folder.
Figure 2: A single-molecule assay for probing the nanomechanics of the RLC complex
- Fig2G: Contains Force(N) vs Extension(m) data using BioLever Long cantilever with data smoothed to 25kHz
- Fig2H: Contains Force(N) vs Extension(m) data collected using BioLever Fast cantilever with data smoothed to 25kHz
Figure 3: Two intermediates revealed when pulling across the LARLC.
- Fig3A: Force_smth(1-4)(N) vs Ext_smth(1-4)(m). Columns contain Force vs Extension data for the 4 curves in SI units. Data smoothed to 2.5kHz using a 2nd-order Savitzky-Golay filter
- Fig3B: Force_smth(1-4)(N) vs time_(1-4)(s). Columns contain Force vs time data for the 4 curves in SI units. Data smoothed to 500Hz using a 2nd-order Savitzky-Golay filter
- Fig3C: Force(N), Force_smth(N) vs time(s). Columns contain Force data smoothed to 25kHz and 500Hz in newtons and time in seconds
Figure 4: Structural insights derived from coarse-grained simulations.
Fig4D: WLC curves fitted the computational data
- WLC_F_N(pN) vs WLC_Ext_N(nm). Contains the WLC force vs extension curve for the native state.
- WLC_F_O(pN) vs WLC_Ext_O(nm). Contains the WLC force vs extension curve for the open state.
- WLC_F_I2(pN) vs WLC_Ext_I2(nm). Contains the WLC force vs extension curve for the I2 state.
- WLC_F_U(pN) vs WLC_Ext_U(nm). Contains the WLC force vs extension curve for the unfolded state.
- Segments of computational Force vs Extension from 10 combined curves to which the WLC model is fitted.
- F_N(pN) vs Ext_N(nm). Segment to which the WLC model is fitted to the native state.
- F_O(pN) vs Ext_O(nm). Segment to which the WLC model is fitted in the open state.
- F_I2(pN) vs Ext_I2(nm). Segment to which the WLC model is fitted to the I2 state.
- F_none(pN) vs Ext_none(nm). The segment that is not included in the WLC fits due to helix unwinding.
- F_U(pN) vs Ext_U(nm). Segment to which the WLC model is fitted in the unfolded state.
Fig4G: Experimental force extension curve with computationally predicted delta L values.
- Force_smth(N) vs Ext_smth(m). Experimental Force vs extension curve, data smoothed to 1.5kHz using a 2nd-order Savitzky-Golay filter.
- WLC_F_N(N) vs WLC_Ext_N(nm). WLC force vs extension curve for the native state.
- WLC_F_O(N) vs WLC_Ext_O(nm). WLC force vs extension curve for open state.
- WLC_F_I2(N) vs WLC_Ext_I2(nm). WLC force vs extension curve for I2 state.
- WLC_F_U(N) vs WLC_Ext_U(nm). WLC force vs extension curve for unfolded state.
Figure 5: Changes in structural contracts during ankle opening and initial EF-hand release.
- Fig5A: Force(pN) vs time(us). Force vs time data for a replica showing back-and-forth transitions.
- Fig5B: Q_H1,Q_Ankle,Q_H2 vs time(us). Averaged fraction of interal native contacts in H1, Ankle, and H2, respectively, vs time
- Fig5C: Q_H1-RLC,Q_Ankle-RLC,Q_H2-RLC vs time(us). Averaged fraction of interal native contacts between H1 and RLC, Ankle and RLC, H2 and RLC, respectively, vs time
- Fig5D: Q_CALLI vs time(us). Fraction of native contacts in CALLI vs time.
Figure 6: CALLI stabilized the acute ankle
- Fig6C: deltaN_F_smth(N) vs deltaN_Ext_smth(m). Force vs extension curve for the delta N construct. Data smoothed to 3.3kHz using a 2nd-order Savitzky-Golay filter.
WT_F_smth(N) vs WT_Ext_smth(m). Force extension curve for WT construct. Data smoothed to 3.3kHz using a 2nd-order Savitzky-Golay filter.
- Fig6D: WT,deltaN Cumulative probability vs Force(pN)
- Fig6E: WT_mean_F(pN),deltaN_meanF(pN) are the mean forces ankle opening forces for WT and deltaN, respectively.
WT_SEM_F(pN),deltaN_SEM_F(pN) are sem for the WT and deltaN, respectively.
Figure 7: Dynamic force spectroscopy analysis of ankle opening
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Fig7A: MeanF(N) vs MeanLR(N/s). Mean ankle opening force vs mean loading rate in SI units for increasing velocities.
SemF(N), SemLR(N) for the corresponding means.
FitF(N) vs FitLR(N/s). Bell model fit to the meanFs
V1F(N) vs V1LR(N/s). Unfolding forces vs loading rate at the corresponding forces at velocity=100nm/s
V2F(N) vs V2LR(N/s). Unfolding forces vs loading rate at the corresponding forces at velocity=200nm/s
V3F(N) vs V3LR(N/s). Unfolding forces vs loading rate at the corresponding forces at velocity=600nm/s
V4F(N) vs V4LR(N/s). Unfolding forces vs loading rate at the corresponding forces at velocity=1000nm/s
V5F(N) vs V5LR(N/s). Unfolding forces vs loading rate at the corresponding forces at velocity=1600nm/s
V6F(N) vs V6LR(N/s). Unfolding forces vs loading rate at the corresponding forces at velocity=3000nm/s
V7F(N) vs V7LR(N/s). Unfolding forces vs loading rate at the corresponding forces at velocity=10000nm/s
Figure 8: Different unfolding pathways for different pulling geometries.
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Fig8B: F(1-6)(N) vs Ext(1-6)(m). Force vs extention curves for pulling across the RLC. Data smoothed to 1kHz using a 2nd-order Savitzky-Golay filter.
WLC_N_F(N) vs WLC_N_Ext(m). WLC fits the native state.
WLC_I_F(N) vs WLC_I_Ext(m). WLC curve with average increase in contour length for intermediate state.
Fig8Cbottom: F(1-12)(pN) vs Ext(1-12)(nm). Force vs extension curves from simulations for 12 replicas in which the EFn unfolded first when pulling across RLC.
WLC_N_F(pN) vs WLC_N_E(nm). WLC fit to the combined data from the native state of the 12 replicas.
WLC_I_F(pN) vs WLC_I_E(nm). WLC fit to the combined data from the intermediate state of the 12 replicas.
Fig8Ctop: F(1-5)(pN) vs Ext(1-5)(nm). Force vs extension curves from simulations for 5 replicas for which the EFc unfolded first when pulling across RLC.
WLC_N_F(pN) vs WLC_N_E(nm). WLC fit to the combined data from the native state of the 5 replicas.
WLC_I_F(pN) vs WLC_I_E(nm). WLC fit to the combined data from the intermediate state of the 5 replicas.
- Fig8G: F(1-6)(N) vs Ext(1-6)(m). Force vs extention curves for pulling across the RLC complex. Data smoothed to 1kHz using a 2nd-order Savitzky-Golay filter.
WLC_N_F(N) vs WLC_N_Ext(m). WLC fits the native state.
WLC_I_F(N) vs WLC_I_Ext(m). WLC curve with average increase in contour length for the intermediate state.
Fig8H: F(1-18)(pN) vs Ext(1-18)(nm). Force vs extension curves from simulations for 18 replicas in which the EFn unfolded first.
WLC_N_F(pN) vs WLC_N_E(nm). WLC fit to the combined data from the native state of the 18 replicas.
WLC_I_F(pN) vs WLC_I_E(nm). WLC fit to the high force region in the combined data from the intermediate state of the 18 replicas.
Supplementary Information:
Figure S2:Characterization of FIB-modified ultrashort BioLever Fast and uncoated BioLever Long AFM cantilevers in liquid at 100 nm above the surface, determined from their thermal motion.
- FigS2A: FP_fast(pN) vs FP_fast_t(s). Force precision vs averaging time for FIB-modified BioLever Fast cantilever.
FP_Long(pN) vs FP_long_t(s). Force precision vs averaging time for an uncoated BioLever Long cantilever
FigS2B: Autocorrelation_fast vs fast_t(s). Autocorrelation vs lagtime for a BioLever Fast cantilever.
Autocorrelation_long vs long_t(s). Autocorrelation vs lagtime for an uncoated BioLever Long cantilever.
Figure S3: Varied unfolding trajectories.
FigS3A: F_smth(N) vs Ext_smth(m). Force vs extension curve showing no open state. Data smoothed to 1.5kHz using a 2nd-order Savitzky-Golay filter.
WLC_'X'F(N) vs WLC'X'_Ext(m). WLC curves with computationally predicted delta Ls. Where 'X':{N (native), O (open), I2 (intermediate), and U (unfolded)}
FigS3B: F_smth(N) vs Ext_smth(m). Force vs extension curve showing gradual transition from I2 to U. Data smoothed to 1.5kHz using a 2nd-order Savitzky-Golay filter.
WLC_'X'F(N) vs WLC'X'_Ext(m). WLC curves with computationally predicted delta Ls. Where 'X':{N (native), O (open), I2 (intermediate), and U (unfolded)}
FigS3C: F_smth(N) vs Ext_smth(m). Force vs extension curve showing gradual transition from I2 to U. Data smoothed to 1.5kHz using a 2nd-order Savitzky-Golay filter.
WLC_'X'F(N) vs WLC'X'_Ext(m). WLC curves with computationally predicted delta Ls. Where 'X':{N (native), O (open), I2 (intermediate), and U (unfolded)}
FigS3D: F_smth(N) vs Ext_smth(m). Force vs extension curve showing gradual transition from I2 to U. Data smoothed to 1.5kHz using a 2nd-order Savitzky-Golay filter.
WLC_'X'F(N) vs WLC'X'_Ext(m). WLC curves with computationally predicted delta Ls. Where 'X':{N (native), O (open), I2 (intermediate), and U (unfolded)}
Figure S6: Histogram of absolute total contour length (Lo) of the construct and change in contour length (delta Lo) due to GB1 unfolding.
Fig6A: N vs bins(nm). Number of molecules vs bins of total contour length.
- Fig. 6B: N vs bins(nm). Number of molecules vs bins of delta Lo
Figure S8: Coarse-grained Brownian dynamics simulations of the deltaN construct.
- FigS8A: WT_F(pN) vs WT_Ext(nm). Force vs extension curve from WT simulation.
deltaN_F(pN) vs deltaN_E(nm). Force vs extension curve from deltaN simulation
- FigS8B: Angle(degrees),Angle_smooth(degrees) vs time(us). Angle vs time curves for the delta N construct.
FigS8C: WT_mean_F(pN),deltaN_meanF(pN) are the mean forces ankle opening forces for WT and deltaN simulations, respectively.
WT_SEM_F(pN),deltaN_SEM_F(pN) are sem for the WT and deltaN simulations, respectively.
Figure S9: Data-acquisition protocol for the dynamic force spectrum.
- FigS9A: Attach_F(N) vs Attach_t(s). Variation of force during the attachment phase of the protocol as a function of time.
Ramp2surf(N) vs Ramp2surf_t(s). Variation of force as a function of time during the ramp back to the surface after successful attachment.
F(1-7)(N) vs t(1-7)(s). Force variation as a function of time during stretch and relax cycles. The 1s dwell starts when the cantilever reaches the surface. Full 1s dwell data not recorded.
Detach_F(N) vs Detach_t(s). Force variation as a function of time during the detachment phase of the protocol.
- FigS9B: Attach_Z(m) vs Attach_t(s). Distance between surface and base of the cantilever during the attachment phase of the protocol as a function of time
Ramp2surf_Z(m) vs Ramp2surf_t(s). Distance between surface and base of the cantilever as a function of time, during the ramp back to the surface after successful attachment.
Z(1-7)(m) vs t(1-7)(s). Distance between surface and base of the cantilever as a function of time during stretch and relax cycles. Full 1s dwell data not recorded.
Detach_Z(m) vs Detach_t(s). Distance between surface and base of the cantilever as a function of time during the detachment phase of the protocol.
Figure S10: Seven consecutive force-extension curves.
- FigS10A-G: F(1-7)(N) vs Ext(1-7)(m). Force extension curves with varying velocities were taken during the stretch and relax cycles.
- Figure S11: Comparison between initial and repeated ankle-opening force.
FigS11: InitialOpeningMean(pN), RepeatedOpeningMean(pN) are the means of initial ankle opening forces from different molecules and repeatedly from the same molecule, respectively.
InitialOpeningSem(pN) and RepeatedOpeningSem(pN) are SEM for the initial ankle opening forces and repeated ankle opening forces, respectively.
Figure S12: Deducing angular rotation to the transition state using the coarse-grained simulation.
- FigS12A-F: Ext(nm), Ext_smth(nm) vs time(us). Variation of extension as a function of time, with 3.78 nm as the horizontal dashed line.
Angle(degrees), Angle(degrees) vs time(us). Variation of angle between H1 and H2 as a function of time.
Qcalli, Qcalli_smth vs time(us). Variation of the fraction of native contact in calli as a function of time.
*D-F: the zoomed-in data is taken from A-C.
Figure S13: Histogram of ankle angles under force in the native and open state.
- FigS13: N_RF vs N_bins(degrees). Relative frequency of angles in the native state vs their bins.
O_lowF_RF vs O_lowF_bins(degrees). Relative frequency of angles in the open force under low force and their bins.
O_highF_RF vs O_highF_bins(degrees). Relative frequency of angles in the open force under high force and their bins.
Figure S14: Deducing the fraction of persistent native contacts (Qankle) that stabilizes the native state against ankle opening at the predicted transition state.
- FigS14A: Angle(degrees),Angle_smth(degrees) vs time(us). Variation in ankle angle as a function of time.
- FigS14B-C: Qankle,Qankle_smth vs time(us). Variation in the fraction of native contacts that stabilize the native state as a function of time.
*C: Zoomed-in data is taken from B
Figure S15: Unfolding force for the EFc domain when pulling across RLC and its mechanical dissociation when pulling across RLC in simulations.
- FigS15: RLC_pull_EFcForce_mean(pN), LA_pull_EFcForce_mean(pN) are mean forces for unfolding of EFc when pulling along RLC and its dissociation force when pulling along LA, respectively.
RLC_pull_EFcForce_sem(pN), LA_pull_EFcForce_sem(pN) are sem for the (RLC pulling) EFc unfolding force and (LA pulling) EFc dissciation force, respectively.
Figure S16: Procedure depicting removal of optical-interference artifact (OIA).
- Fig16A: deltaZmeasured_ret(m) vs Zpzt_ret(m). deltaZ measured as a function of Zpzt (stage movement) during retract.
deltaZmeasured_app(m) vs Zpzt_app(m). deltaZ measured as a function of Zpzt (stage movement) during approach.
deltaZmeasured_app_seg(m) vs Zpzt_app_seg(m). Segment of the approach curve corresponding to the region where the protein is attached in the retract curve.
deltaZmeasured_ret_seg(m) vs Zpzt_ret_seg(m). Segment of the retract curve in the region where the protein has detached.
OIA_fity(m) vs OIA_fitx(m). OIA fits the combined approach and retract segments.
- Fig16B: Force(N) vs Extension(m). The force extension curve after removal of the OIA.
- Fig16C: deltaZmeasured_app(m) vs Zpzt_app(m). deltaZ measured as a function of Zpzt (stage movement) during approach.
deltaZmeasured_ret(m) vs Zpzt_ret(m). deltaZ measured as a function of Zpzt (stage movement) during retract with protein not attached.
OIAFity(m) vs OIAFitx(m). OIA fits the approach curve.
- Fig16D: Force_smth(N) vs Extension_smth(m). Force extension curve after removal of OIA from the retract curve in C. Data smoothed to 100Hz using 2nd order savitzky-golay filter.