Subnanometre enzyme mechanics probed by single-molecule force spectroscopy

Pelz, Benjamin; Žoldák, Gabriel; Zeller, Fabian; Zacharias, Martin; Rief, Matthias

doi:10.1038/ncomms10848

Cited by 63 publications

(74 citation statements)

References 42 publications

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“…The instrument for optical trapping and single-molecule fluorescence was adapted from that previously described (52,53). Single-molecule fluorescence capability was built using a 488-nm, <100-ps excitation laser (BDL-488-SMC; Becker & Hickl) operated at 20 MHz and modulated electronically using the same field-programmable gate array (FPGA) used to steer the optical trap and acquire data (NI PCI-7833R 3M; National Instruments).…”

Section: Methodsmentioning

confidence: 99%

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Ganim

Rief

2017

Proc. Natl. Acad. Sci. U.S.A.

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Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.optical tweezers | protein folding | fluorescent protein | mechanoswitch G reen fluorescent protein (GFP) is a 27-kDa β-barrel protein with an intrinsic chromophore. (1, 2) It is widely used in imaging applications that rely on its structural stability [to thermal and high-pressure unfolding (3), in fusion constructs (4), and to circular permutation (5)] or its optical response to environment, such as in biosensors for force transduction (6-9), calcium concentration (10), protease activity (11), and pH (12, 13). Understanding the relationship between protein structure and fluorescence is essential for these applications. Photophysical properties of the chromophore are sensitive to its hydrogen bonding, solvation, isomerization state, and binding-pocket structure. (1,2,14). It is known that fluorescence is quenched upon denaturation and that several intermediates have been observed in folding experiments and simulations (3,(15)(16)(17)(18)(19)(20)(21), although it has not been possible to definitively probe the fluorescence properties of partially structured intermediates in isolation from the native state. Speculation exists regarding whether single-molecule photobleaching can be reversed by unfolding and refolding the protein (22). It is not known whether tension on the native state alters fluorescence in similarity to the compressive, pressure-induced elastic effect (3) and whether its emission can be reversibly mechanically switched. In this study, we address these questions by making an explicit connection between structure and fluorescence, using single-molecule methods.Single-molecule experiments are widely used to reveal distributions in biophysical structure and kinetics that underlie ensemble-averaged properties. Single-molecule manipulation using force experiments such as optical tweezers can be used to observe states nominally at negligible concentration in a bulk distribution (23)(24)(25). Single-molecule fluorescence experiments have been used to probe the ...

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Section: Methodsmentioning

confidence: 99%

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Ganim

Rief

2017

Proc. Natl. Acad. Sci. U.S.A.

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Section: Enzymes As Motorsmentioning

confidence: 99%

Enzyme Catalysis To Power Micro/Nanomachines

et al. 2016

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Enzymes play a crucial role in many biological processes which require harnessing and converting free chemical energy into kinetic forces in order to accomplish tasks. Enzymes are considered to be molecular machines, not only because of their capability of energy conversion in biological systems but also because enzymatic catalysis can result in enhanced diffusion of enzymes at a molecular level. Enlightened by nature’s design of biological machinery, researchers have investigated various types of synthetic micro/nanomachines by using enzymatic reactions to achieve self-propulsion of micro/nanoarchitectures. Yet, the mechanism of motion is still under debate in current literature. Versatile proof-of-concept applications of these enzyme-powered micro/nanodevices have been recently demonstrated. In this review, we focus on discussing enzymes not only as stochastic swimmers but also as nanoengines to power self-propelled synthetic motors. We present an overview on different enzyme-powered micro/nanomachines, the current debate on their motion mechanism, methods to provide motion and speed control, and an outlook of the future potentials of this multidisciplinary field.

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“…However, such analogies are often misleading because boundaries between independently stable subdomains cannot often be determined from structures, owing to the high cooperativity of protein folding and structural transitions. Single-molecule protein nanomechanics have emerged as a tool to force biomolecules through their conformational space and, hence, identify hinges, breaking points, and mechanically stable subdomains (1)(2)(3).…”

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confidence: 99%

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

Mandal

Merz

Buchsteiner

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α-and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.laser trapping | parallel pathways | elasticity | force | protein extension W hen looking at protein structures at atomic resolution, it is often tempting to use macroscopic mechanical analogies to describe their function as molecular machines. However, such analogies are often misleading because boundaries between independently stable subdomains cannot often be determined from structures, owing to the high cooperativity of protein folding and structural transitions. Single-molecule protein nanomechanics have emerged as a tool to force biomolecules through their conformational space and, hence, identify hinges, breaking points, and mechanically stable subdomains (1-3).A prominent example of a protein machine undergoing large conformational change during its functional cycle is the ATP-regulated Hsp70 chaperone DnaK-a central molecular chaperone of the protein quality control network in a cell (4-6). Once ATP is bound to the nucleotide binding domain (NBD, blue-yellow; Fig. 1A) of DnaK, the initially closed substrate binding domain (SBD) opens its binding cleft by engaging the β-subdomain to the NBD. In doing so, it undergoes a dramatic ∼10 Å displacement of its lid subdomain ( Fig. 1A; refs. 7 and 8) to allow exchange of substrates (9). Several crystal structures of the isolated SBD (in which the NBD is absent) have been solved (10-15). In these structures, the absence or presence of peptide clients or nonnatural ligands induce no significant structural changes in the closed conformation. There is no indication in the crystal structures of the huge conformational change of the lid domain of the SBD, seen in the ATP form of the full-length two-domain DnaK. Therefore, the large conformational change of the SBD is only observed in the two-domain DnaK after ATP binding. Thus, although the crystal structures provide us with valuable insights into the 3D arrangement of individual atoms, the thermodynamic and mechanical stability of individual substructures are difficult to predict based on this information alone. Here, we ask how the large ATP-induced changes of the SBD, as seen in the two-domain DnaK, are mirrored in the subdomain integrity and nano...

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Subnanometre enzyme mechanics probed by single-molecule force spectroscopy

Cited by 63 publications

References 42 publications

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Enzyme Catalysis To Power Micro/Nanomachines

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

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