Facile tethering of stable and unstable proteins for optical tweezers experiments

Maciuba, Kevin; Zhang, Fan; Kaiser, Christian

doi:10.1016/j.bpj.2021.05.003

Cited by 20 publications

(16 citation statements)

References 55 publications

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“…Oligonucleotides were then hybridized with an overhang presented in longer DNA handles [125]. Other chemical couplings have been developed (reviewed, for example, here [126]), including click chemistry, unnatural amino acids and others [127][128][129][130]. Using different coupling strategies enables the attachment of linkers of different mechanical elastic properties, which may affect the quality of the signal.…”

Section: Advances In Single-molecule Force Spectroscopy Of Proteinsmentioning

confidence: 99%

Single-molecule mechanical studies of chaperones and their clients

Rief

2022

Biophysics Reviews

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Single-molecule force spectroscopy provides access to the mechanics of biomolecules. Recently, magnetic and laser optical tweezers were applied in the studies of chaperones and their interaction with protein clients. Various aspects of the chaperone–client interactions can be revealed based on the mechanical probing strategies. First, when a chaperone is probed under load, one can examine the inner workings of the chaperone while it interacts with and works on the client protein. Second, when protein clients are probed under load, the action of chaperones on folding clients can be studied in great detail. Such client folding studies have given direct access to observing actions of chaperones in real-time, like foldase, unfoldase, and holdase activity. In this review, we introduce the various single molecule mechanical techniques and summarize recent single molecule mechanical studies on heat shock proteins, chaperone-mediated folding on the ribosome, SNARE folding, and studies of chaperones involved in the folding of membrane proteins. An outlook on significant future developments is given.

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Section: Advances In Single-molecule Force Spectroscopy Of Proteinsmentioning

confidence: 99%

Single-molecule mechanical studies of chaperones and their clients

Rief

2022

Biophysics Reviews

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“…To apply mechanical force to a target protein in magnetic or optical tweezers [4][5][6][7] , we tether two protein positions, e.g., the N-and C-termini, to solid supports of a micron-sized bead and the sample chamber surface (or another bead), respectively. Rapid noncovalent tethering has been widely used, such as the binding between digoxigenin (dig) and antidigoxigenin (antidig), 6xHis tag and Ni-NTA, and biotin and streptavidin [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . However, the noncovalent tethers are dissociated in tens-of-pN force ranges 23,24 , restricting the utilization of the tweezer methods.…”

Section: Introductionmentioning

confidence: 99%

Robust single membrane protein tweezers

Kim

Lee

Wijesinghe

et al. 2023

Preprint

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Single-molecule tweezers, such as magnetic tweezers, are powerful mechanical manipulation tools that can probe nm-scale structural changes in a single membrane protein under force. However, the weak molecular tethers used in the tweezers limit a long time, repetitive mechanical manipulation because of their force-induced bond breakage. Here, using the metal-free click chemistry of dibenzocyclooctyne (DBCO) cycloaddition and the rapid, strong binding of traptavidin to dual biotins (2xbiotin), we developed robust single-molecule tweezers that can perform thousands of force applications on a single membrane protein. By applying up to 50 pN for each cycle, which is sufficiently high for most biological processes, we were able to observe repetitive forced unfolding for a designer membrane protein up to approximately 1000 times on average. Monte Carlo simulations showed that the average error of the unfolding kinetic values rapidly decays to 1.8% at 200-time pulling, indicating that our method can quickly produce reliable statistics. The approach established here is also applicable to highly polar DNA molecules, permitting the nanomechanical manipulation of diverse biomolecular systems.

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“…In practice, these leashes may consist in peptidic chains [1, 2, 4, 22, 23], single DNA strands [24, 25, 26], or double DNA strands, with [15, 27, 28] or without nicks [29, 30, 31]. While peptidic linkers require to design and produce a specific fusion protein, nucleic acids based structures offer more modularity when combined with state-of-the-art DNA-protein coupling strategies [20, 30, 32, 33, 34, 35, 36, 37]. As a matter of fact, the latter kind of scaffold have already been used for non-covalent bond characterization on various SMFS setups: OT [26, 24, 25, 15, 14], MT [30, 25, 29, 31], CFM [15], and LFC [28].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

Wang

Valotteau

Aimard

et al. 2022

Preprint

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Single-molecule data are of great significance in biology, chemistry, and medicine. However, experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are needed. Acoustic force spectroscopy (AFS) is an emerging manipulation technique which generates acoustic waves to apply force in parallel on a large population of microbeads tethered to a surface. We have exploited this configuration on a recently developed modular Junctured-DNA (J-DNA) scaffold designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measured its unbinding kinetics under force at the single-bond level. Special effort was made in analyzing the data in order to identify potential pitfalls. We established a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy for measuring the force dependent rupture of a single domain antibody with its antigen. We get a good agreement with standard measurement at zero force. Our technique offers single molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

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Facile tethering of stable and unstable proteins for optical tweezers experiments

Cited by 20 publications

References 55 publications

Single-molecule mechanical studies of chaperones and their clients

Single-molecule mechanical studies of chaperones and their clients

Robust single membrane protein tweezers

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

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