Pulling geometry defines the mechanical resistance of a β-sheet protein

Brockwell, David J.; Paci, Emanuele; Zinober, Rebecca C.; Beddard, Godfrey S.; Olmsted, Peter D.; Smith, D. A.; Perham, Richard N.; Radford, Sheena E.

doi:10.1038/nsb968

Cited by 367 publications

(350 citation statements)

References 43 publications

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“…Indeed, it is well recognized that the features of the energy landscapes of proteins that determine the folding pathways and kinetic barriers are controlled by a subtle balance between native topology and energetics that stabilize the folded structure (3)(4)(5). The present and previous studies show that the energy landscape can presumably be manipulated by using cross-link mutants or by merely changing the points of force application that alters the direction along which strain propagates (16,27,35), and hence the unfolding routes in the rugged energy landscape (Fig. 6).…”

Section: Discussionmentioning

confidence: 95%

“…In part, the difficulty arises because complex proteins generally fold slowly and tend to aggregate in bulk experiments (8), a problem that can be avoided in mechanical unfolding of single proteins. Indeed, single-molecule mechanical methods have recently provided new possibilities for directly probing the energy landscapes of proteins and RNA because they can monitor the population of partially folded states (9)(10)(11)(12)(13)(14)(15)(16). In addition, these experiments can explore regions of the energy landscape that are inaccessible in conventional experiments, thus providing a complete picture of the folding process (17)(18)(19)(20).…”

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confidence: 99%

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Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations

Mickler

Dima

Dietz

et al. 2007

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

153

201

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Nanomanipulation of biomolecules by using single-molecule methods and computer simulations has made it possible to visualize the energy landscape of biomolecules and the structures that are sampled during the folding process. We use simulations and single-molecule force spectroscopy to map the complex energy landscape of GFP that is used as a marker in cell biology and biotechnology. By engineering internal disulfide bonds at selected positions in the GFP structure, mechanical unfolding routes are precisely controlled, thus allowing us to infer features of the energy landscape of the wild-type GFP. To elucidate the structures of the unfolding pathways and reveal the multiple unfolding routes, the experimental results are complemented with simulations of a self-organized polymer (SOP) model of GFP. The SOP representation of proteins, which is a coarse-grained description of biomolecules, allows us to perform forced-induced simulations at loading rates and time scales that closely match those used in atomic force microscopy experiments. By using the combined approach, we show that forced unfolding of GFP involves a bifurcation in the pathways to the stretched state. After detachment of an N-terminal ␣-helix, unfolding proceeds along two distinct pathways. In the dominant pathway, unfolding starts from the detachment of the primary N-terminal ␤-strand, while in the minor pathway rupture of the last, C-terminal ␤-strand initiates the unfolding process. The combined approach has allowed us to map the features of the complex energy landscape of GFP including a characterization of the structures, albeit at a coarse-grained level, of the three metastable intermediates.AFM experiments ͉ coarse-grained simulations ͉ cross-link mutants ͉ pathway bifurcation ͉ plasticity of energy landscape P rotein structures, which are astounding examples of selforganization in living systems, reach their folded states by navigating through a rugged energy landscape. Considerable progress has been made in understanding the folding mechanisms of small, single-domain proteins by using a combination of theory and experiments (1-5). Folding of many of these proteins can be approximately described as being two-state-like, that is, their energy landscape does not exhibit pronounced local minima corresponding to partially folded or misfolded structures. However, the folding energy landscapes of larger proteins, with complex topology, can be difficult to characterize because of the presence of multiple metastable intermediate structures (6,7). A detailed characterization of the structures of the intermediate states and the associated energetics is a challenge for the experimentalist. In part, the difficulty arises because complex proteins generally fold slowly and tend to aggregate in bulk experiments (8), a problem that can be avoided in mechanical unfolding of single proteins. Indeed, single-molecule mechanical methods have recently provided new possibilities for directly probing the energy landscapes of proteins and RNA because they can monito...

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Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 99%

Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations

Mickler

Dima

Dietz

et al. 2007

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

153

201

View full text Add to dashboard Cite

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“…The native state is mechanically resistant because C is pulled longitudinally, so that several bonds must break simultaneously. Once C is gone, nothing keeps the ␤-hairpin B from unzipping, one bond at a time; unzipping requires less force than separation by longitudinal pulling (33,34). Similarly, for the three structures A, D, and E, which form the typical intermediate state, it is clear that the ␤-hairpin E is protected by D (see Fig.…”

Section: Summary and Discussionmentioning

confidence: 98%

Dissecting the mechanical unfolding of ubiquitin

Irbäck

Mitternacht

Mohanty

2005

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

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The unfolding behavior of ubiquitin under the influence of a stretching force recently was investigated experimentally by single-molecule constant-force methods. Many observed unfolding traces had a simple two-state character, whereas others showed clear evidence of intermediate states. Here, we use Monte Carlo simulations to investigate the force-induced unfolding of ubiquitin at the atomic level. In agreement with experimental data, we find that the unfolding process can occur either in a single step or through intermediate states. In addition to this randomness, we find that many quantities, such as the frequency of occurrence of intermediates, show a clear systematic dependence on the strength of the applied force. Despite this diversity, one common feature can be identified in the simulated unfolding events, which is the order in which the secondary-structure elements break. This order is the same in two-and three-state events and at the different forces studied. The observed order remains to be verified experimentally but appears physically reasonable. all-atom model ͉ force-induced unfolding ͉ Monte Carlo simulation T he 76-residue protein ubiquitin fulfills many important regulatory functions in eukaryotic cells through its covalent attachment to other proteins (1, 2). In many cases, the ubiquitin tag consists of a chain of ubiquitin domains (polyubiquitin), which is formed by linkages between an exposed lysine side chain of the last ubiquitin of a growing chain and the C terminus of a new ubiquitin. The fate of a polyubiquitin-tagged protein depends on the linkage. For example, Lys-48-C-linked polyubiquitin marks the protein substrate for proteasomal degradation (3).Recently, Fernandez and coworkers (4-7) and Chyan et al. (8) investigated the mechanical properties of polyubiquitin by single-molecule force spectroscopy. It was shown that Lys-48-Clinked as well as end-to-end (N-C)-linked polyubiquitin can withstand a stretching force; the average unfolding force was 85 pN for Lys-48-C linkage and Ϸ200 pN for N-C linkage (4). In these experiments, the polyubiquitin chains were pulled with a constant velocity. In another experiment on N-C-linked polyubiquitin, the stretching force was kept constant (6). At constant force, the fraction of unfolded ubiquitin domains was found to show an approximately single-exponential time dependence, as expected if the unfolding of individual domains is a simple Markovian two-state process. Nevertheless, the unfolding of individual domains sometimes occurred through intermediate states. The precise nature of these different unfolding pathways, including the structure of the intermediate states, remains to be determined. We stress that these intermediates are states along forced unfolding trajectories. To what extent there are significant folding intermediates for small proteins is a debated (9, 10), but different, issue. Here, we use Monte Carlo (MC) simulations to examine the unfolding of ubiquitin under a constant stretching force in atomic detail. Our calculations are b...

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“…A hierarchy of unfolding energies of the unfolding crystals may be simply due to inhomogeneity effects of the crystal domains [14,35,56], showing variable bond-breaking barriers [33] possibly owing to interfacial energy effects [57]. Another important effect is anisotropicity of the crystals with respect to the force direction-different paths in the wiggly energy landscape lead to different unfolding energy barriers [58]-so that different unfolding forces can be induced by variable crystal orientations in the macromolecule.…”

Section: Unfolding Energy Hierarchymentioning

confidence: 99%

An energetic model for macromolecules unfolding in stretching experiments

Tommasi

Millardi

Puglisi

et al. 2013

J. R. Soc. Interface.

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We propose a simple approach, based on the minimization of the total (entropic plus unfolding) energy of a two-state system, to describe the unfolding of multidomain macromolecules (proteins, silks, polysaccharides, nanopolymers). The model is fully analytical and enlightens the role of the different energetic components regulating the unfolding evolution. As an explicit example, we compare the analytical results with a titin atomic force microscopy stretch-induced unfolding experiment showing the ability of the model to quantitatively reproduce the experimental behaviour. In the thermodynamic limit, the sawtooth force-elongation unfolding curve degenerates to a constant force unfolding plateau.

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Pulling geometry defines the mechanical resistance of a β-sheet protein

Cited by 367 publications

References 43 publications

Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations

Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations

Dissecting the mechanical unfolding of ubiquitin

An energetic model for macromolecules unfolding in stretching experiments

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