Single-molecule approaches to characterizing kinetics of biomolecular interactions

Oijen, Antoine M. van

doi:10.1016/j.copbio.2010.10.002

Cited by 113 publications

(113 citation statements)

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“…In addition, iSCAT sensing can be used to visualize the motion of proteins (ref. 36) and monitor the association and dissociation kinetics of biomolecules and study their cooperative interactions 37 because it does not suffer from photobleaching. The sensitivity of iSCAT in our experiment was determined by the pixel well depth of the camera.…”

Section: Discussionmentioning

confidence: 99%

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

2014

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Detection of single analyte molecules without the use of any label would improve the sensitivity of current biosensors by orders of magnitude to the ultimate graininess of biological matter. Over two decades, scientists have succeeded in pushing the limits of optical detection to single molecules using fluorescence. However, restrictions in photophysics and labelling protocols make this technique less attractive for biosensing. Recently, mechanisms based on vibrational spectroscopy, photothermal detection, plasmonics and microcavities have been explored for fluorescence-free detection of single biomolecules. Here, we show that interferometric detection of scattering (iSCAT) can achieve this goal in a direct and label-free fashion. In particular, we demonstrate detection of cancer marker proteins in buffer solution and in the presence of other abundant proteins. Furthermore, we present super-resolution imaging of protein binding with nanometer localization precision. The ease of iSCAT instrumentation promises a breakthrough for label-free studies of interactions involving proteins and other small biomolecules. T here are more than 2,000 proteins secreted by the human body at concentrations that are below the detection limit of the current commercial biosensors 1 . To achieve a sufficient sensitivity for early and robust diagnosis of health hazards such as cancer and lethal infectious diseases, scientists have explored a fury of strategies based on mechanical 2 , electrochemical 3 and optical 4,5 interactions. The ultimate performance would be to count the analyte molecules one at a time. Furthermore, a practical biosensor would ideally detect the molecule of interest directly and without the need for labelling, amplification or sophisticated architectures.A well-established approach for label-free detection is via the vibrational signatures of molecules, for example via Raman spectroscopy. This method has an exquisite selectivity, but singlemolecule sensitivity has only been reached by enhancing the Raman cross-section in the near field of surfaces 6 or tips 7 . A common alternative strategy relies on the detection of the refractive index change due to analyte binding. The oldest and commercially available implementation of this technique records the shift of the plasmon resonance of a gold-coated prism 8 , whereby the specificity and selectivity are provided by surface chemistry 9 . While submonolayer detection is readily achievable in such a device, a single molecule does not manifest a significant change in the refractive index of the sensor medium. To increase the sensitivity, confined modes of plasmonic nanostructures [10][11][12] and dielectric microresonators 13,14 have been investigated, but these techniques are intrinsically limited. First, there is a compromise between the size of the sensor active area and its sensitivity. Higher sensitivity comes through confined intensity distributions and, thus, at the cost of more restricted active area, fewer binding receptors and thinner functionalization 15 . Se...

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Section: Discussionmentioning

confidence: 99%

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

2014

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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).…”

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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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“…The canonical description of enzymatic inhibition received much exposure 1,2,11 , but even at the level of bulk reactions its many limitations have already been pointed out 12 . Moreover, and despite rapid advancements in the study of uninhibited enzymatic reactions on the singlemolecule level, the study of inhibited reactions has barely made progress in this direction and is still based, by and large, on what is known in bulk.Single molecule approaches revolutionized our understanding of enzymatic catalysis 13,14 . Early work demonstrated that at the single molecule level enzymatic catalysis is inherently stochastic 15,16 , and that one often needs to go beyond the common Markovian description to adequately account for the observed kinetics 17,18,19,20 .…”

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confidence: 99%

“…Single molecule approaches revolutionized our understanding of enzymatic catalysis 13,14 . Early work demonstrated that at the single molecule level enzymatic catalysis is inherently stochastic 15,16 , and that one often needs to go beyond the common Markovian description to adequately account for the observed kinetics 17,18,19,20 .…”

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confidence: 99%

“…Single molecule approaches revolutionized our understanding of enzymatic catalysis 13,14 . Early work demonstrated that at the single molecule level enzymatic catalysis is inherently stochastic enzyme kinetics, including the widespread applicability of the Michaelis-Menten equation and its insensitivity to microscopic details, were discovered 21,22,23,24,25,26,27 ; and the study of enzymatic reactions under force has shed new light on the mechanics of the catalytic process 28,29,30,31,32 .…”

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confidence: 99%

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Single-molecule theory of enzymatic inhibition predicts the emergence of inhibitor-activator duality

Robin

Reuveni

Urbakh

2016

Preprint

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The classical theory of enzymatic inhibition aims to quantitatively describe the effect of certain molecules-called inhibitors-on the progression of enzymatic reactions, but growing signs indicate that it must be revised to keep pace with the single-molecule revolution that is sweeping through the sciences. Here, we take the single enzyme perspective and rebuild the theory of enzymatic inhibition from the bottom up. We find that accounting for multiconformational enzyme structure and intrinsic randomness cannot undermine the validity of classical results in the case of competitive inhibition; but that it should strongly change our view on the uncompetitive and mixed modes of inhibition. There, stochastic fluctuations on the single-enzyme level could give rise to inhibitor-activator duality-a phenomenon in which, under some conditions, the introduction of a molecule whose binding shuts down enzymatic catalysis will counter intuitively work to facilitate product formation.We state-in terms of experimentally measurable quantities-a mathematical condition for the emergence of inhibitor-activator duality, and propose that it could explain why certain molecules that act as inhibitors when substrate concentrations are high elicit a nonmonotonic dose response when substrate concentrations are low. The fundamental and practical implications of our findings are thoroughly discussed.Enzymes spin the wheel of life by catalyzing a myriad of chemical reactions central to the growth, development, and metabolism of all living organisms 1,2 . Without enzymes, essential processes would progress so slowly that life would virtually grind to a halt; and some enzymatic reactions are so critical that inhibiting them may result in death. Enzymatic inhibitors could thus be potent poisons 3,4 but could also be used as antibiotics 5,6 and drugs to treat other forms of disease 7,8 . Inhibitors have additional commercial uses 9,10 , but the fundamental principles which govern their interaction with enzymes are not always understood in full, and have yet ceased to fascinate those interested in the basic aspects of enzyme science. The canonical description of enzymatic inhibition received much exposure 1,2,11 , but even at the level of bulk reactions its many limitations have already been pointed out 12 . Moreover, and despite rapid advancements in the study of uninhibited enzymatic reactions on the singlemolecule level, the study of inhibited reactions has barely made progress in this direction and is still based, by and large, on what is known in bulk.Single molecule approaches revolutionized our understanding of enzymatic catalysis 13,14 . Early work demonstrated that at the single molecule level enzymatic catalysis is inherently stochastic 15,16 , and that one often needs to go beyond the common Markovian description to adequately account for the observed kinetics 17,18,19,20 . Universal aspects of stochastic . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for ...

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Single-molecule approaches to characterizing kinetics of biomolecular interactions

Cited by 113 publications

References 56 publications

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

Single-molecule theory of enzymatic inhibition predicts the emergence of inhibitor-activator duality

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