Direct Observation of the Reversible Two‐State Unfolding and Refolding of an α/β Protein by Single‐Molecule Atomic Force Microscopy

He, Chengzhi; Hu, Chunguang; Hu, Xiaodong; Xiao, Adam; Perkins, Thomas T.; Li, Hongbin

doi:10.1002/anie.201502938

Cited by 55 publications

(63 citation statements)

References 40 publications

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“…31 Analysis of a single individual polyprotein yielded k o = 0.03 ± 0.02 s −1 and Δ x ‡ = 4.2 ± 0.4 Å, where k o is the off-rate at zero force, and Δ x ‡ is the distance to the unfolding-transition state. These parameters are in quantitative agreement with the recently reported values (0.04 s −1 , and 4.2 Å).…”

Section: Resultsmentioning

confidence: 97%

“…Analysis of the force-extension curves yielded a change in contour length (Δ L = 17.4 nm) that matched the literature values for NuG2 (Δ L ≈ 17.6 nm). 30,31 We then repeated the measurement at different pulling velocities to acquire a dynamic force spectrum (Figure 5c) at one surface location (Figure 5d) for a total of 357 unfolding events in 2 h.…”

Section: Resultsmentioning

confidence: 99%

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Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation

Walder

LeBlanc²,

Patten

et al. 2017

J. Am. Chem. Soc.

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Atomic-force-microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α–helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation

Walder

LeBlanc²,

Patten

et al. 2017

J. Am. Chem. Soc.

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“…NuG2 is a computationally designed fast folding variant of the protein GB1, and its mechanical unfolding has been studied using AFM techniques . Here we carried out OT experiments to compare the SMFS results on a single protein versus its polyprotein.…”

Section: Figurementioning

confidence: 99%

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

et al. 2016

Angew Chem Int Ed

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Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2) as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2) lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments.

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“…The combination of subnanometer spatial resolution and piconewton force sensitivity enables not only the measurement of the single‐molecule mechanics but also the extraction of kinetic parameters . The single‐molecule mechanics can also provide the structural information while measuring the length of the unfolded chain . Much has been learned about the working mechanism of molecular motor and the adsorption behavior at solid‐liquid interfaces, as well as the force‐induced conformation transition of individual polymer chain .…”

Section: Introductionmentioning

confidence: 99%

Force‐induced melting of a single polyethylene oxide chain from single crystal: Molecular behavior and influencing factors

Song

Yang

Jiang

et al. 2019

Polymer Crystallization

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Force‐induced melting plays a key role in defining the mechanical strength and toughness of semicrystalline polymer materials. To understand its molecular mechanism, the direct measurement and monitoring of force‐induced melting process at the single‐molecule level are necessary. Here, the force‐induced melting of a single polyethylene oxide (PEO) chain from the single crystal has been investigated by using atomic force microscopy‐based single‐molecule force spectroscopy. Both the constant‐speed and constant‐force experiments show that PEO folds are unfolded predominantly one by one during the force‐induced melting process. The mechanical stability of PEO fold is confirmed to increase significantly with the increasing interfacial tension of polymer solvent and the thickness of crystal. The results obtained here provide new concepts in tuning the nanomechanical properties of crystalline polymer materials.

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Direct Observation of the Reversible Two‐State Unfolding and Refolding of an α/β Protein by Single‐Molecule Atomic Force Microscopy

Cited by 55 publications

References 40 publications

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation

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

Force‐induced melting of a single polyethylene oxide chain from single crystal: Molecular behavior and influencing factors

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