Single‐Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2

Lei, Hong; He, Chengzhi; Hu, Chunguang; Li, Jinliang; Hu, Xiaodong; Hu, Xiaotang; Li, Hongbin

doi:10.1002/ange.201610648

Cited by 10 publications

(6 citation statements)

References 37 publications

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“…The mechanical unfolding of NuG2 is characterized by a contour length increment (ΔL c ) of ∼18 nm, 28,29 and unfolding forces of ∼30 pN at a pulling speed of 50 nm/s. 30 The observation of two unfolding events of NuG2 in a given force−distance curve ensures that the force− extension curve contains the unfolding signature of RTX. 25 Figure 2 shows two pairs of representative stretching− relaxation force−distance curves of apo-NuG2-RTX-NuG2, in which only two unfolding and refolding events of NuG2 with ΔL c of 18 nm were present.…”

Section: ■ Resultsmentioning

confidence: 99%

Single Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of the Bacterial Toxin Protein RTX

Wang

Gao

2019

J. Am. Chem. Soc.

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The efficient translocation of the bacterial toxin adenylate cyclase toxin (CyaA) from the bacterial cytosol to the extracellular environment by the type 1 secretion system (T1SS) is essential for the toxin to function. To understand the molecular features that are responsible for the efficient translocation of CyaA, here we used optical tweezers to investigate the mechanical properties and conformational dynamics of the RTX domain of CyaA at the single molecule level. Our results revealed that apo-RTX behaves like an ideal random coil. This property allows the T1SS to translocate RTX without overcoming the enthalpic resistance. In contrast, the folded holo-RTX is mechancially stable, and its folding occurs in a vectorial, cotranslocational fashion starting from its C-terminus. Moreover, our results showed that the folding of holo-RTX generates a stretching force, which can further facilitate the translocation of RTX. Our results highlight the important role played by the Ca2+-triggered folding of RTX in the translocation of RTX and provide mechanistic insights into the mechanical design that governs the efficient translocation of RTX.

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Section: ■ Resultsmentioning

confidence: 99%

Single Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of the Bacterial Toxin Protein RTX

Wang

Gao

2019

J. Am. Chem. Soc.

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“…Optical trapping experiments were carried out on a Mini-Tweezers setup, which was described previously. 22,23 The construction of double-stranded DNA (dsDNA) handles and protein-DNA chimera was carried out following well-established protocols, as described. A streptavidinmodified polystyrene bead (with a diameter of ∼1 μm, purchased from Spherotech) was sucked by a micropipette tip, and an antidigoxigenin bead (with a diameter of ∼2 μm, purchased from Spherotech) sparsely covered with protein-DNA chimera was trapped by a laser beam and brought into close proximity to build a bead-DNA-protein dumbbell.…”

Section: Optical Trapping Experimentsmentioning

confidence: 99%

Harvesting Mechanical Work From Folding-Based Protein Engines: From Single-Molecule Mechanochemical Cycles to Macroscopic Devices

Wang

2019

CCS Chem

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Mechanochemical coupling cycles underlie the work-generation mechanisms of biological systems and are realized by highly regulated conformational changes of the protein machineries. However, it has been challenging to utilize protein conformational changes to do mechanical work at the macroscopic level in biomaterials, and it remains elusive to construct macroscopic mechanochemical devices based on molecular-level mechanochemical coupling systems. Here, the authors demonstrate that protein folding can be utilized to realize protein’s mechanochemical cycles at both single-molecule and macroscopic levels. Our results demonstrate, for the first time, the successful harnessing of mechanical work generated by protein folding in a macroscopic protein hydrogel device, and the work generated by protein folding compares favorably with the energy output of molecular motors. Our work bridges a gap between single-molecule and macroscopic levels, and paves the way to utilizing proteins as building blocks to design protein-based artificial muscles and soft actuators.

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“…2D). These results further highlight that the different monomers forming a polyprotein chain fold independently, implying that the folding dynamics of an individual monomer is independent of the composition of the rest of the multidomain construct 24,25 . Most importantly, our results reveal that the dynamic change in compliance of a multi-protein construct can be used as a direct proxy to identify the number of folded (and unfolded) modules within an actively folding homopolyprotein at a given time.…”

Section: Protein Stiffness Evolution In a Dynamically Folding Polypromentioning

confidence: 70%

Molecular fluctuations as a ruler of force-induced protein conformations

Stannard

Mora

Beedle

et al. 2020

Preprint

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Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis can probe protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically-stable states scales with the protein's contour length, in agreement with the sequence-independent FJC model of polymer physics. Our findings will help probe the conformational dynamics of proteins exposed to mechanical force at high resolution, of central importance in mechanosensing and mechanotransduction.

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Single‐Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2

Abstract: Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: http://dx.

Cited by 10 publications

References 37 publications

Single Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of the Bacterial Toxin Protein RTX

Single Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of the Bacterial Toxin Protein RTX

Harvesting Mechanical Work From Folding-Based Protein Engines: From Single-Molecule Mechanochemical Cycles to Macroscopic Devices

Molecular fluctuations as a ruler of force-induced protein conformations

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