Nanovesicle Trapping for Studying Weak Protein Interactions by Single-Molecule FRET

Benítez, Jaime J.; Keller, Aaron M.; Chen, Peng

doi:10.1016/s0076-6879(10)72016-4

Cited by 24 publications

(40 citation statements)

References 35 publications

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“…Following a similar reasoning, researchers have been able to capture fluorescently labeled binding partners in small (100-200 nm), surface-tethered lipid nanovesicles and study their interactions through single-molecule FRET measurements (Figure 2b) [51][52][53]54 ]. The volume of a 100-nm vesicle is two orders of magnitude smaller than that of a diffraction-limited volume and thus allows proteins to be visualized at the single-molecule level at much higher effective concentrations than hitherto possible.…”

Section: The Concentration Problemmentioning

confidence: 98%

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Oijen

2011

Current Opinion in Biotechnology

113

111

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Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van Oijen, A. M. (2011). Single-molecule approaches to characterizing kinetics of biomolecular interactions. Current Opinion in Biotechnology, 22(1), 75-80. DOI: 10.101675-80. DOI: 10. /j.copbio.2010 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Available online at www.sciencedirect.com Single-molecule approaches to characterizing kinetics of biomolecular interactions Antoine M van OijenSingle-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of individual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.

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Section: The Concentration Problemmentioning

confidence: 98%

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Oijen

2011

Current Opinion in Biotechnology

113

111

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“…For instance, circular zero-mode waveguides (ZMWs) constrain fluorescent excitation in sub-micron scale chambers, as described in more detail below [3]. Alternatively, fluorescent substrates can be constrained in lipid vesicles, which can either be trapped or surface-tethered, or in trapped droplets suspended in oil [4][5][6]. All of these approaches allow imaging at up to micromolar concentrations by limiting the excitation volume in all three dimensions, which works very well for studying substrates whose dimensions are all sub-micron and that do not undergo micron-scale motions.…”

Section: Introductionmentioning

confidence: 99%

Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC)

et al. 2013

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Abstract:Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.

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“…Single-molecule approaches to biomolecular interaction kinetics (24)(25)(26) allow for direct measurements of binding and unbinding rates (27)(28)(29). In addition, the combination of single-molecule florescence (16,18) with single-molecule force spectroscopy (23, 30) marks itself as an especially promising tool in unbinding studies because the fluorescence readout for catalytic activity is expected to be correlated with selective, force-induced, changes of the activation barrier for unbinding (23,30).…”

mentioning

confidence: 99%

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

Reuveni

Urbakh

Klafter

2014

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

238

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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Nanovesicle Trapping for Studying Weak Protein Interactions by Single-Molecule FRET

Cited by 24 publications

References 35 publications

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC)

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

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