Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy

Pérez-Jiménez, Raúl; Li, Jingyuan; Kosuri, Pallav; Sánchez-Romero, Inmaculada; Wiita, Arun P.; Rodríguez-Larrea, David; Chueca, Ana; Holmgren, Arne; Miranda‐Vizuete, Antonio; Becker, Katja; Cho, Seung-Hyun; Beckwith, Jon; Gelhaye, Éric; Jacquot, Jean‐Pierre; Gaucher, Eric A.; Sanchez-Ruiz, José M.; Berne, B. J.; Fernández, Julio M.

doi:10.1038/nsmb.1627

Cited by 83 publications

(117 citation statements)

References 61 publications

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“…Several studies have shown that when force is applied to the proteins in certain enzyme-substrate complexes, the relaxation is very sensitive to strength of the applied force, sometimes in a nonlinear fashion (4). An analysis of the kind presented here might shed light on this sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Elasticity, structure, and relaxation of extended proteins under force

Stirnemann¹,

Giganti

Fernández

et al. 2013

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

110

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Force spectroscopies have emerged as a powerful and unprecedented tool to study and manipulate biomolecules directly at a molecular level. Usually, protein and DNA behavior under force is described within the framework of the worm-like chain (WLC) model for polymer elasticity. Although it has been surprisingly successful for the interpretation of experimental data, especially at high forces, the WLC model lacks structural and dynamical molecular details associated with protein relaxation under force that are key to the understanding of how force affects protein flexibility and reactivity. We use molecular dynamics simulations of ubiquitin to provide a deeper understanding of protein relaxation under force. We find that the WLC model successfully describes the simulations of ubiquitin, especially at higher forces, and we show how protein flexibility and persistence length, probed in the force regime of the experiments, are related to how specific classes of backbone dihedral angles respond to applied force. Although the WLC model is an average, backbone model, we show how the protein side chains affect the persistence length. Finally, we find that the diffusion coefficient of the protein's end-to-end distance is on the order of 10 8 nm 2 /s, is position and side-chain dependent, but is independent of the length and independent of the applied force, in contrast with other descriptions.internal diffusion | potential of mean force | protein elasticity T he development of single-molecule force spectroscopies [atomic force microscopy (AFM), and optical or magnetic tweezers] in the last two decades has opened a whole new and exciting field (1, 2). It is now possible to manipulate biomolecules directly at a molecular level and to study their behavior under force (3). It is therefore not surprising that these techniques have been applied in a broad gamut of contexts, in particular for proteins. In some cases, including enzyme catalysis (4), protein-ligand interaction (5), or folding and unfolding events (6), force is used as a probe, altering the free-energy landscape and the dynamics of the protein (7), and provides valuable kinetic and mechanistic insights. For other systems, force is of direct biological relevance [e.g., cellular adhesion (8) or muscle elasticity (9)].The elasticity of a polypeptide is typically modeled using the worm-like chain (WLC) (1, 2) model of polymer elasticity. Although this simple model from polymer physics has proven remarkably successful at describing and interpreting experimental data, it lacks molecular details associated with proteins extended under force. However, substrate flexibility to adopt the right geometry is key in many situations, including, e.g., molecular recognition (10) or enzymatic reactivity (11). Therefore, a precise understanding of how force modulates and influences protein flexibility at a single-amino acid level is essential and is not provided by the aforementioned model.From a dynamical perspective, it is still unclear how applied force affects internal diffusion ...

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Section: Discussionmentioning

confidence: 99%

Elasticity, structure, and relaxation of extended proteins under force

Stirnemann¹,

Giganti

Fernández

et al. 2013

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

110

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“…25,26 Most likely, this interface is generally used for specificity in interaction, and possibly also to generate correct alignment of target disulfide bonds with Cys 32, which single-molecule studies suggest requires rearrangement at the interface for formation of the intermolecular disulfide bond intermediate. 27,28 …”

Section: Functional Protein-protein Interfacementioning

confidence: 99%

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

Weichsel¹,

Kem²,

Montfort³

2010

Protein Science

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Thioredoxins reduce disulfide bonds and other thiol modifications in all cells using a CXXC motif. Human thioredoxin 1 is unusual in that it codes for an additional three cysteines in its 105 amino acid sequence, each of which have been implicated in other reductive activities. Cys 62 and Cys 69 are buried in the protein interior and lie at either end of a short helix (helix 3), and yet can disulfide link under oxidizing conditions. Cys 62 is readily S-nitrosated, giving rise to a SNO modification, which is also buried. Here, we present two crystal structures of the C69S/C73S mutant protein under oxidizing (1.5 Å ) and reducing (1.1 Å ) conditions. In the oxidized structure, helix 3 is unraveled and displays a new conformation that is stabilized by a series of new hydrogen bonds and a disulfide link with Cys 62 in a neighboring molecule. The new conformation provides an explanation for how a completely buried residue can participate in SNO exchange reactions.

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“…Interestingly, the hyperbolic dependence of turnover on substrate concentration remains valid even at the single-molecule level (12,14). From a theoretical perspective, this result was initially puzzling but is now considered almost universal in the sense that it can be shown to hold under a wide range of modeling assumptions (20, 21).…”

mentioning

confidence: 93%

“…Recent advancements in single-molecule spectroscopy have gradually made it possible to follow the stochastic activity of individual enzymes over extended periods of time (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Interestingly, the hyperbolic dependence of turnover on substrate concentration remains valid even at the single-molecule level (12,14).…”

mentioning

confidence: 99%

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

Reuveni

Urbakh

Klafter

2014

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

241

218

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The Michaelis-Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover. Surprisingly, this prediction was never tested experimentally nor was it scrutinized using modern theoretical tools. Here we show that unbinding may also speed up enzymatic turnover-turning a spotlight to the fact that its actual role in enzymatic catalysis remains to be determined experimentally. Analytically constructing the unbinding phase space, we identify four distinct categories of unbinding: inhibitory, excitatory, superexcitatory, and restorative. A transition in which the effect of unbinding changes from inhibitory to excitatory as substrate concentrations increase, and an overlooked tradeoff between the speed and efficiency of enzymatic reactions, are naturally unveiled as a result. The theory presented herein motivates, and allows the interpretation of, groundbreaking experiments in which existing single-molecule manipulation techniques will be adapted for the purpose of measuring enzymatic turnover under a controlled variation of unbinding rates. As we hereby show, these experiments will not only shed first light on the role of unbinding but will also allow one to determine the time distribution required for the completion of the catalytic step in isolation from the rest of the enzymatic turnover cycle.single enzyme | enzyme kinetics | renewal theory E very enzymatic reaction is composed out of two basic steps:(i) the reversible binding of a substrate molecule to the enzyme and (ii) a catalytic step which gives rise to the formation of the product (1). Controlled variation of the effective binding rate to a free enzyme is attained by altering the concentration of the substrate. The rate of product formation, also known as the turnover rate, can then be measured as a function of this concentration to show a characteristic hyperbolic dependence (2-5). The Michaelis-Menten equation is used to rationalize this observation and interpret the results (2, 6).Recent advancements in single-molecule spectroscopy have gradually made it possible to follow the stochastic activity of individual enzymes over extended periods of time (7-19). Interestingly, the hyperbolic dependence of turnover on substrate concentration remains valid even at the single-molecule level (12,14). From a theoretical perspective, this result was initially puzzling but is now considered almost universal in the sense that it can be shown to hold under a wide range of modeling assumptions (20, 21). One can thus safely say that the role of binding in Michaelis-Menten enzymatic reactions is well understood. In this paper, we consider the role of unbinding.Rapid advancements in the single-molecule technological front imply that the adaptation of existing single-molecule manipulation techniques (12-14, 18, 22, 23) to allow the measurement of enzymatic turnover under a controlled variation of unbinding rates is now within reach. Single-molecule approaches to biomolecular inter...

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Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy

Cited by 83 publications

References 61 publications

Elasticity, structure, and relaxation of extended proteins under force

Elasticity, structure, and relaxation of extended proteins under force

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

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