Force-Induced Ankle Opening Reveals Mechanical Stabilization of the Ankle of Human β-Cardiac Myosin

ACS Nano · 2026-05-21

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.

A Type III secretion system effector evolved to be mechanically labile and initiate unfolding from the N-terminus

bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-04

Abstract Many Gram-negative pathogens critically depend on the Type III secretion system (T3SS) to inject effector proteins into host cells for colonization. Because the channel of the T3SS is narrow (∼2 nm), effectors must be unfolded for secretion. However, the T3SS cannot unfold mechanically robust substrates (GFP, ubiquitin, and dihydrofolate reductase), severely impairing their secretion. Consistent with this, effectors are exceptionally mechanically labile, unfolding at low forces. Thus, secretion competency is correlated with mechanical properties. Effector sequences have significantly diverged from non-effectors, suggesting that secretion exerts evolutionary pressure selecting mechanical lability. Here, using atomic-force-microscopy–based force spectroscopy, we show that effector NleC is mechanically labile ( F unfold = 13.5 pN at 100 nm/s) and mechanically compliant, as characterized by a large distance to the transition state (Δ x ‡ = 2.7 nm). In contrast, the non-effector homolog protealysin is mechanically stable ( F unfold = 50.7 pN at 100 nm/s) and brittle (Δ x ‡ = 0.7 nm), comparable to proteins known to impair secretion ( F unfold > 80 pN; Δ x ‡ < 0.4 nm). Denaturant-induced unfolding assays demonstrate that effectors exhibit rates typical of their fold, further reinforcing mechanical properties rather than fast unfolding kinetics ( k 0 ) predicts secretion. Steered molecular dynamic simulations revealed NleC unfolding initiates at the N-terminus, consistent with current secretion models, whereas protealysin unfolding initiates at the C-terminus. Notably, the NleC N-terminus is primarily α-helical while non-effector homologs contain β-sheets, which may account for the distinct unfolding pathway. Together, these results support the notion that mechanical lability is an evolved, structurally encoded feature underlying effector secretion. Significance The Type III secretion system (T3SS) delivers effector proteins directly into host cells to promote bacterial colonization. Effectors must be unfolded for secretion, and this particular selective pressure is hypothesized to have driven significant sequence divergence from non-effector proteins. Here, we show that effectors are not characterized by unusually fast unfolding rates. Rather as hypothesized, effector NleC is more mechanically labile than its non-effector homolog, indicating that mechanical lability underlies both effector sequence divergence and T3SS unfolding. Simulations revealed that NleC unfolding initiates via the N-terminus consistent with the current secretion mechanism, while protealysin unfolds from the C-terminus. Together, these results strongly suggest mechanical lability is an evolved property of effectors and provide structural insight into how it is encoded.

Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy

Proceedings of the National Academy of Sciences · 2024-02-07 · 5 citations

Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification. Here, we measured these energetics on the millisecond time scale using atomic-force-microscopy-based single-molecule force spectroscopy. Precisely, timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8-amino-acid region of bR's G-helix. These dynamics changed when the EF-helix pair moved ~9 Å away from this end of the G helix during the "open" portion of bR's photocycle. In ~60% of the data, we observed abrupt light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pH-dependence of this destabilization were consistent with prior measurements of bR's open phase. The frequency of light-induced destabilization increased with the duration of illumination and was dramatically reduced in the triple mutant (D96G/F171C/F219L) thought to trap bR in its open phase. In the other ~40% of the data, photoexcitation unexpectedly stabilized a longer-lived putative misfolded state. Through this work, we establish a general single-molecule force spectroscopy approach for measuring ligand-induced energetics and lifetimes in membrane proteins.

Force-activated DNA substrates for single-molecule studies

2023-10-05

Optical traps are widely used to study nucleic-acid-processing enzymes. To investigate these enzymes, we developed “force-activated” DNA substrates that contain a pair of nicks, allowing displacement of single-stranded DNA when pulled into DNA’s overstretching transition. We designed these substrates to include DNA hairpins and are using them to investigate the mechanism of E. coli RecQ helicase, an enzyme that unwinds double-stranded DNA. Force-activated DNA substrates also have the potential to provide an easy-to-use intrinsic force standard at ~15 pN, suitable for all three major force spectroscopy modalities (i.e., optical traps, magnetic tweezers, and AFM).

Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay

Methods in molecular biology · 2022-01-01 · 1 citations

Type III secretion system effector proteins are mechanically labile

Proceedings of the National Academy of Sciences · 2021 · 54 citations

• Cell biology
• Biology
• Chemistry

< 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

Proceedings of the National Academy of Sciences · 2021 · 20 citations

• Chemistry
• Crystallography
• Biophysics

for a fully folded membrane protein embedded in its native bilayer.

Investigation of a Possible Novel Genetic Mutation in Brangus Calves with Proportionate Dwarfism

2021-03-04

Data Collection Methodology: Genome-wide association studies (GWAS) were conducted in GenABEL (Aulchenko et al. 2007) with sex as a covariate and accounting for population stratification in the sample using a relationship matrix and/or principle components; the means most appropriate and thus the model assumed for various iterations of the data (e.g., with and without additional samples) was determined by the estimate of lambda, a measure of genomic inflation. For an additional means to conduct association analyses and to allow for investigation of regions of significance from the GWAS studies, DNA from four affected calves and their dams as well as from the semen of the sire was isolated and whole-genome sequenced. The resulting sequence was aligned to the UCD-ARS1.2 reference genome with BWA-MEM (Li 2013) and variants identified with freebayes (Garrison & Marth 2012). The functional impact of each variant was predicted using SNPEff (Cingolani et al. 2012). Expected Findings: Mapping and variant calling of WGS from the nine Brangus individuals resulted in 6.9 million variants for evaluation. Assuming a genetic cause of this condition is rare across breeds, WGS data from Heaton et al. (2016) representing over 100 individuals of over 20 breeds (including 6 Angus, 5 Brangus, and 5 Brahman) and that of the 12 other calves sequenced by our lab were utilized to remove common variants (found at >5% frequency). This reduced the data set to be more manageable with 51,460 candidate variants fitting a recessive mode of inheritance. 43,000 of the total variants remaining after filtering were predicted to have either a “high” or “moderate” impact on gene function. The WGS data were utilized to investigate the regions identified by GWAS of possible interest as well as to investigate possible variants in genes previously implicated in dwarfism in cattle and other species (Table 3). In regions of interest, variants were first examined to find any that may fit a recessive mode of inheritance (heterozygous in each parent but homozygous in the affected calves). As no clear candidate were resolved, variants having a predicted impact were also considered allowing for some error in phenotype assignment (e.g. only 3⁄4 calves affected). The possibility of a dominant trait resulting from mosaicism of the sire, in which case he may show no symptoms but pass this defect to a proportion of his offspring, was also considered by examining loci where the sire and calves are all heterozygous while the dams do not have the variant. In all evaluations, the position of each variant in the genome (e.g., intergenic, intronic, exonic) and predicted function was noted to prioritize variants with possible functional relevance.

Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

Proceedings of the National Academy of Sciences · 2021 · 31 citations

• Chemistry
• Chemical physics
• Crystallography

) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Correcting molecular transition rates measured by single-molecule force spectroscopy for limited temporal resolution

Physical review. E · 2020-08-05 · 12 citations

Equilibrium free-energy-landscape parameters governing biomolecular folding can be determined from nonequilibrium force-induced unfolding by measuring the rates k for transitioning back and forth between states as a function of force F. However, bias in the observed forward and reverse rates is introduced by limited effective temporal resolution, which includes the mechanical response time of the force probe and any smoothing used to improve the signal-to-noise ratio. Here we use simulations to characterize this bias, which is most prevalent when the ratio of forward and reverse rates is far from unity. We find deviations in k(F) at high rates, due to unobserved transitions from short- to long-lived states, and at low rates, due to the corresponding unobserved transitions from long- to short-lived states. These missing events introduce erroneous curvature in log(k) vs F that leads to incorrect landscape parameter determination. To correct the measured k(F), we derive a pair of model-independent analytical formulas. The first correction accounts for unobserved transitions from short- to long-lived states, but does surprisingly little to correct the erroneous energy-landscape parameters. Only by subsequently applying the second formula, which corrects the corresponding reverse process, do we recover the expected k(F) and energy-landscape quantities. Going forward, these corrections should be applied to transition-rate data whenever the highest measured rate is not at least an order of magnitude slower than the effective temporal resolution.