Free Energy Landscape of Type III Fibronectin Domain with Identified Intermediate State and Hierarchical Symmetry

Hao Sun, Zilong Guo, Haiyan Hong, Zhuwei Zhang, Yuhang Zhang, Yang Wang, Shimin Le, and Hu Chen

Phys. Rev. Lett. 131, 218402 – Published 22 November 2023

Abstract

The tenth domain of type III fibronectin ( ${FNIII}_{10}$ ) mediates cell adhesion to the extracellular matrix. Despite its structural similarity to immunoglobulin domains, ${FNIII}_{10}$ exhibits unique unfolding behaviors. We employed magnetic tweezers to investigate the unfolding and folding dynamics of ${FNIII}_{10}$ under physiological forces (4–50 pN). Our results showed that ${FNIII}_{10}$ follows a consistent transition pathway with an intermediate state characterized by detached A and G $β$ strands. We determined the folding free energies and all force-dependent transition rates of ${FNIII}_{10}$ and found that both unfolding rates from the native state to the intermediate state and from the intermediate state to the unfolded state deviate from Bell’s model. We constructed a quantitative free energy landscape with well-defined traps and barriers that exhibits a hierarchical symmetrical pattern. Our findings provide a comprehensive understanding of ${FNIII}_{10}$ conformational dynamics and demonstrate how free energy landscape of multistate biomolecules can be precisely mapped, illuminating the relationship between thermal stability, intermediate states, and folding rates in protein folding.

Received 17 December 2022
Accepted 23 October 2023

DOI:https://doi.org/10.1103/PhysRevLett.131.218402

Physics Subject Headings (PhySH)

Bimolecular conformation Protein folding Protein unfolding

Proteins

Single molecule techniques

Physics of Living SystemsInterdisciplinary PhysicsStatistical Physics & ThermodynamicsNonlinear DynamicsCondensed Matter, Materials & Applied PhysicsPhysics Education ResearchPolymers & Soft Matter

Authors & Affiliations

Hao Sun ^1,2, Zilong Guo², Haiyan Hong¹, Zhuwei Zhang^1,2, Yuhang Zhang¹, Yang Wang², Shimin Le¹, and Hu Chen ^1,2,*

¹Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics, Xiamen University, Xiamen 361005, China
²Center of Biomedical Physics, Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou 325000, China

^*chenhu@xmu.edu.cn

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Vol. 131, Iss. 21 — 24 November 2023

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Images

Figure 1
Single-molecule force spectroscopy measurements with MT. (a) Schematic representation of protein construct in MT (not drawn in proportion). The reference bead is used to remove the drift. (b) ${FNIII}_{10}$ (amino acids (a.a.) 1–94, protein data bank:1ttf) and (c) ${FNIII}_{10}^{Δ AG}$ (a.a. 13–80) unfolding, SpyTag–SpyCatcher (un)zipping, and I27 unfolding events were recorded sequentially when force increases from 1 to 80 pN with loading rate of $0.5 pN / s$ . Insets show the native structure of ${FNIII}_{10}$ (b) and ${FNIII}_{10}^{Δ AG}$ (c). Raw data in (b) and (c) recorded at a sampling rate of 200 Hz (gray) are smoothed using 50-ms time window (black). Uncertainty of force is estimated as 5%.
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Figure 1
Single-molecule force spectroscopy measurements with MT. (a) Schematic representation of protein construct in MT (not drawn in proportion). The reference bead is used to remove the drift. (b) ${FNIII}_{10}$ (amino acids (a.a.) 1–94, protein data bank:1ttf) and (c) ${FNIII}_{10}^{Δ AG}$ (a.a. 13–80) unfolding, SpyTag–SpyCatcher (un)zipping, and I27 unfolding events were recorded sequentially when force increases from 1 to 80 pN with loading rate of $0.5 pN / s$ . Insets show the native structure of ${FNIII}_{10}$ (b) and ${FNIII}_{10}^{Δ AG}$ (c). Raw data in (b) and (c) recorded at a sampling rate of 200 Hz (gray) are smoothed using 50-ms time window (black). Uncertainty of force is estimated as 5%.
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Figure 2
Force-dependent folding and unfolding transitions of ${FNIII}_{10}$ . (a) Typical folding and unfolding processes of ${FNIII}_{10}$ were recorded by MT. Specifically, one-step folding processes of ${FNIII}_{10}$ were observed when force dropped from 12 to 6 pN. Apparently, different unfolding pathways were observed when force jumps from 1 pN to 9, 15, and 40 pN. (b) At constant forces of 7 and 7.5 pN, extension traces show the equilibrium folding and unfolding transitions between N and U states of ${FNIII}_{10}$ . Raw data in (a) and (b) with a sampling rate of 200 Hz (gray) are smoothed using 50-ms time window (black). (c) Average folding rates ( $k^{UN}$ ) at forces smaller than 6.5 pN, equilibrium folding and unfolding rates ( $k^{UN}$ and $k^{NU}$ ) from 6.85 to 7.5 pN (enlarged figure), and transition rates ( $k^{NI}$ , $k^{IN}$ , $k^{IU}$ , and $k_{u}^{app}$ ) at forces greater than 8 pN of ${FNIII}_{10}$ are acquired from more than four independent tethers. $k^{NI} (F)$ , $k^{IU} (F)$ , and $k_{u}^{app} (F)$ show nonlinear behavior deviating from the prediction of Bell’s model. Theoretical unfolding rate from N to U ( $k_{u}^{theor}$ ) calculated from Eq. (2) agrees well with experimental $k_{u}^{app}$ . The solid lines (the same color as corresponding symbols) are fitting results by Bell’s model in different force ranges. Uncertainty of force is estimated as 5%.
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Figure 2
Force-dependent folding and unfolding transitions of ${FNIII}_{10}$ . (a) Typical folding and unfolding processes of ${FNIII}_{10}$ were recorded by MT. Specifically, one-step folding processes of ${FNIII}_{10}$ were observed when force dropped from 12 to 6 pN. Apparently, different unfolding pathways were observed when force jumps from 1 pN to 9, 15, and 40 pN. (b) At constant forces of 7 and 7.5 pN, extension traces show the equilibrium folding and unfolding transitions between N and U states of ${FNIII}_{10}$ . Raw data in (a) and (b) with a sampling rate of 200 Hz (gray) are smoothed using 50-ms time window (black). (c) Average folding rates ( $k^{UN}$ ) at forces smaller than 6.5 pN, equilibrium folding and unfolding rates ( $k^{UN}$ and $k^{NU}$ ) from 6.85 to 7.5 pN (enlarged figure), and transition rates ( $k^{NI}$ , $k^{IN}$ , $k^{IU}$ , and $k_{u}^{app}$ ) at forces greater than 8 pN of ${FNIII}_{10}$ are acquired from more than four independent tethers. $k^{NI} (F)$ , $k^{IU} (F)$ , and $k_{u}^{app} (F)$ show nonlinear behavior deviating from the prediction of Bell’s model. Theoretical unfolding rate from N to U ( $k_{u}^{theor}$ ) calculated from Eq. (2) agrees well with experimental $k_{u}^{app}$ . The solid lines (the same color as corresponding symbols) are fitting results by Bell’s model in different force ranges. Uncertainty of force is estimated as 5%.
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Figure 3
Force-dependent folding and unfolding dynamics of ${FNIII}_{10}^{Δ AG}$ and comparison with ${FNIII}_{10}$ . (a) Schematic representation of protein construct of $AviTag − FH 1 − {FNIII}_{10}^{Δ AG} − FH 1 − SpyTag$ in MT. (b) Typical unfolding and folding processes of ${FNIII}_{10}^{Δ AG}$ were recorded in force-jump experiments. High forces (8 and 10 pN) allow ${FNIII}_{10}^{Δ AG}$ to unfold, and low forces (5 and 6 pN) ensure to observe folding transition of ${FNIII}_{10}^{Δ AG}$ . Raw data with a sampling rate of 200 Hz (gray) are smoothed using 250-ms time window (black). (c) Force-dependent folding and unfolding rates of ${FNIII}_{10}^{Δ AG}$ were summarized from force-jump measurements. $k_{u}$ of ${FNIII}_{10}^{Δ AG}$ agrees with $k^{IU}$ of ${FNIII}_{10}$ in the force range from 8 to 15 pN. $k_{f}$ of ${FNIII}_{10}^{Δ AG}$ , $k^{UN} (F)$ of ${FNIII}_{10}$ , and $k_{f}^{theor} (F)$ calculated from Eq. (3) are consistent with each other. The solid lines (the same color as the corresponding experimental data points) are the fitting results based on Bell’s model in different force ranges. Uncertainty of force is estimated to be 5%.
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Figure 4
The free energy landscape of ${FNIII}_{10}$ constructed by the information of the force sensitivity of the transition rates. Insets show the conformations of N, I, and U states of ${FNIII}_{10}$ , respectively.
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