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Enhanced statistical sampling reveals microscopic complexity in the talin mechanosensor folding energy landscape

Nature Physics volume 19, pages 52–60 (2023)Cite this article

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Abstract

Statistical mechanics can describe the major conformational ensembles determining the equilibrium free-energy landscape of a folding protein. The challenge is to capture the full repertoire of low-occurrence conformations separated by high kinetic barriers that define complex landscapes. Computationally, enhanced sampling methods accelerate the exploration of molecular rare events. However, accessing the entire protein’s conformational space in equilibrium experiments requires technological developments to enable extended observation times. We used single-molecule magnetic tweezers to capture over a million individual transitions as a single talin protein unfolds and refolds under force in equilibrium. When observed at classically probed timescales, talin folds in an apparently uncomplicated two-state manner. As the sampling time extends from minutes to days, the underlying energy landscape exhibits gradually larger signatures of complexity, involving a finite number of well-defined rare conformations. Fluctuation analysis allows us to propose plausible structures of each low-probability conformational state. The physiological relevance of each distinct conformation can be connected to the binding of the cytoskeletal protein vinculin, suggesting an extra layer of complexity in talin-mediated mechanotransduction. More generally, our experiments directly test the fundamental notion that equilibrium dynamics depend on the observation timescale.

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Fig. 1: Enhanced experimental sampling of R3IVVI’s conformational space reveals low-probability states.
Fig. 2: Mechanical force rescues R3IVVI from the rare, low-probability states by bringing it back to its native folding dynamics.
Fig. 3: A proline switch regulates the mechanical response of talin R3IVVI (state (ii)).
Fig. 4: Fluctuation analysis of R3IVVI dynamics fingerprints its wide conformational repertoire, enabling to suggest potential compatible protein structures defining each rare state.
Fig. 5: Conformational space of the talin R3IVVI domain represented as a kinetic network measured at F0.5.
Fig. 6: Probing full-length vinculin’s ability to bind the rare states of R3IVVI.

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Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.

Code availability

The code is available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported in part by the Francis Crick Institute that receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002) and the Wellcome Trust (FC001002). R.T.-R. is the recipient of a King’s Prize Fellowship. O.M. was funded by the Swiss National Foundation grant (310030_207453). This work was supported by the European Commission (Mechanocontrol, grant agreement 731957BBSRC), BBSRC sLoLa (BB/V003518/1), Leverhulme Trust Research Leadership Award (RL-2016-015), Wellcome Trust Investigator Award (212218/Z/18/Z) and Royal Society Wolfson Fellowship (RSWF/R3/183006), to S.G.-M.

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Author notes

  1. These authors contributed equally: Rafael Tapia-Rojo, Marc Mora.

Authors and Affiliations

  1. Single Molecule Mechanobiology Laboratory, The Francis Crick Institute, London, UK

    Rafael Tapia-Rojo, Marc Mora, Stephanie Board, Jane Walker & Sergi Garcia-Manyes

  2. Department of Physics, Randall Centre for Cell and Molecular Biophysics, Centre for the Physical Science of Life and London Centre for Nanotechnology, King’s College London, London, UK

    Rafael Tapia-Rojo, Marc Mora, Stephanie Board, Jane Walker & Sergi Garcia-Manyes

  3. Department of Biochemistry, Zurich University, Zurich, Switzerland

    Rajaa Boujemaa-Paterski & Ohad Medalia

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Contributions

R.T.-R., M.M. and S.G.-M. designed the research. R.T.-R. and M.M. conducted the single-molecule mechanical experiments and analysed the data. S.B. and J.W. expressed and purified the protein constructs. R.B.-P. and O.M. expressed and purified the full-length vinculin. R.T.-R., M.M. and S.G.-M. wrote the paper. All the authors contributed to revising and editing the manuscript.

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Correspondence to Rafael Tapia-Rojo, Marc Mora or Sergi Garcia-Manyes.

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Extended data

Extended Data Fig. 1 Gallery of selected magnetic tweezers recordings of R3IVVI at F0.5.

Each protein spontaneously visits the different identified states (i-vi), highlighted in the insets. The recordings are smoothed with a 101-points Savitzky-Golay algorithm, and unedited (no drift correction). The occasional spikes correspond to floating debris that interferes with the image analysis of the reference or the magnetic bead, but do not affect the intrinsic folding dynamics of the protein.

Extended Data Fig. 2 Gallery of magnetic tweezers recordings showing that a high force pulse rescues R3IVVI from each trapped state back to its native folding dynamics.

State (iii) could not be probed, as it is too short-lived to be rescued with force.

Extended Data Fig. 3 Force dependency of R3IVVI with Pro881 in the trans state (ii).

(A) Dynamics of R3IVVI at F0.5 undergoing a spontaneous cis-trans isomerization of P881. The trans-state is probed at three different forces, showing two-state dynamics with a lower mechanical stability and faster folding/unfolding kinetics. After a few seconds, the cis-state is spontaneously recovered, and R3IVVI goes back to its native folding dynamics. (B) Population of the folded form in the cis-state (grey) and trans-state (red). Cis-trans isomerization of Pro881 shifts the mechanical stability of R3IVVI by ~1 pN.

Extended Data Fig. 4 Force dependency of state (iv).

R3IVVI spontaneously falls into state (iv), characterized by a shorter end-to-end extension (~3 nm shorter than the unfolded state). Exploring forces between 6 and 10 pN shows that this state lacks any internal dynamics (inset).

Extended Data Fig. 5 Force dependency of state (v).

(A) Magnetic tweezers recording of R3IVVI spontaneously falling into state (v). Subsequently, the dynamics of state (v) are measured between 4 pN and 11 pN, allowing one to monitor the relative population of the three conformations. (B) Detail of the recording in (A), which shows the three conformations, L1, L2, and L3. (C) Relative population of conformations L1, L2, and L3 as a function of force. Compared to the canonical native folding dynamics of R3IVVI (i) which exhibits a very sharp force dependency, state (v) shows dynamic transitions between 5 pN and 11 pN.

Extended Data Fig. 6 Force dependency of state (vi).

(A) Magnetic tweezers recording of R3IVVI spontaneously falling into state (vi). Subsequently, the dynamics of state (vi) are measured between 4 pN and 12 pN, allowing us to monitor the relative population of the three conformations. (B) Detail of the recording in (A), which shows the transitions between conformations M1 and M2. (C) Relative population of conformations, M1 and M2, as a function of force.

Extended Data Fig. 7 Unedited 58 hours-long recording of a single protein L at 7.4 pN.

Protein L transitions in equilibrium between the folded and unfolded conformations in a two-state manner, without showing any off-pathway state.

Extended Data Fig. 8 The native (un)folding dynamics in state (i) is reminiscent of a two-state behaviour with our instrumental time resolution.

(A) Raw magnetic tweezers recording showing native unfolding and refolding transitions within state (i) (squared regions). (B) Detail of a native unfolding (left) and refolding (right) transition path, sampled at ~1,500 Hz. (C) Histogram of the trajectory of the reference bead after filtering low-frequency components arising from local drift (blue line). Being firmly attached to the surface, the spread in the vertical position of the reference bead arises mostly due to artifacts in the image analysis algorithm that influence the experimental estimation of the molecular extension. The data are well-fit by a Gaussian (black dotted line) with σ=0.85 nm, which we use as the point-spread function to reconstruct the free energy landscape following a deconvolution procedure. (D) Free energy landscape of talin R3IVVI in state (i) at F0.5. The free energy landscape was reconstructed from >10,000 native (un)folding transitions following a deconvolution procedure (Jansson algorithm, Woodside et al., Science, 2006) using a point-spread function estimated from the trajectory of the reference bead (panel C). The dynamics within state (i) are two-state, as the landscape displays two free energy basins with a single free energy barrier of ~4kT separating them.

Extended Data Table 1 Conformational properties of the different configurational states of the talin R3IVVI domain
Full size table
Extended Data Table 2 Kinetic analysis of average transition times between the different conformations
Full size table

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Tapia-Rojo, R., Mora, M., Board, S. et al. Enhanced statistical sampling reveals microscopic complexity in the talin mechanosensor folding energy landscape. Nat. Phys. 19, 52–60 (2023). https://doi.org/10.1038/s41567-022-01808-4

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