Hardware-based anti-Brownian electrokinetic trap (ABEL trap) for single molecules: control loop simulations and application to ATP binding stoichiometry in multi-subunit enzymes

Jiang, Yan; Wang, Quan; Cohen, Adam E.; Douglas, Nick; Frydman, Judith; Moerner, W. E.

doi:10.1117/12.798093

Cited by 23 publications

(22 citation statements)

References 26 publications

(41 reference statements)

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“…Real-time feedback provides an alternate strategy, and has been used to trap single atoms in vacuum (2). The anti-Brownian electrokinetic (ABEL) trap uses feedback to suppress Brownian motion in solution and can confine particles as small as the 800 kDa complex of the chaperonin GroEL (3)(4)(5). A 104 kDa protein, allophycocyanin, was recently studied in an ABEL trap in which the viscosity was increased with 50% glycerol to slow the Brownian motion (6).…”

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confidence: 99%

Electrokinetic trapping at the one nanometer limit

Fields

Cohen

2011

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

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Anti-Brownian electrokinetic traps have been used to trap and study the free-solution dynamics of large protein complexes and long chains of DNA. Small molecules in solution have thus far proved too mobile to trap by any means. Here we explore the ultimate limits on trapping single molecules. We developed a feedback-based anti-Brownian electrokinetic trap in which classical thermal noise is compensated to the maximal extent allowed by quantum measurement noise. We trapped single fluorophores with a molecular weight of <1 kDa and a hydrodynamic radius of 6.7 Å for longer than one second, in aqueous buffer at room temperature. This achievement represents an 800-fold decrease in the mass of objects trapped in solution, and opens the possibility to trap and manipulate any soluble molecule that can be fluorescently labeled. To illustrate the use of this trap, we studied the binding of unlabeled RecA to fluorescently labeled single-stranded DNA. Binding of RecA induced changes in the DNA diffusion coefficient, electrophoretic mobility, and brightness, all of which were measured simultaneously and on a molecule-by-molecule basis. This device greatly extends the size range of molecules that can be studied by room temperature feedback trapping, and opens the door to further studies of the binding of unmodified proteins to DNA in free solution.A longstanding challenge in single-molecule spectroscopy has been to observe a small molecule in solution for an extended time, without surface tethering or other mechanical immobilization. Stable observation becomes more difficult as the particle decreases in size, because smaller objects diffuse more quickly, in accordance with the Stokes-Einstein relation. Gold nanoparticles as small as 18 nm in diameter, corresponding to a mass of 35 MDa, have been trapped using laser tweezers (1). Below this size laser tweezers fail because the trapping force is proportional to the volume of the trapped object. Real-time feedback provides an alternate strategy, and has been used to trap single atoms in vacuum (2). The anti-Brownian electrokinetic (ABEL) trap uses feedback to suppress Brownian motion in solution and can confine particles as small as the 800 kDa complex of the chaperonin GroEL (3-5). A 104 kDa protein, allophycocyanin, was recently studied in an ABEL trap in which the viscosity was increased with 50% glycerol to slow the Brownian motion (6). Past attempts to trap small-molecule fluorophores in aqueous solution resulted in transient confinement, but not stable trapping (3). Small-molecule fluorophores are the tiniest objects that one can conceive of trapping in aqueous solution. If a particular fluorophore can be trapped, then so too can any molecule to which it is attached.Laser tweezers and the ABEL trap both confine small objects in solution, but the two technologies enable different kinds of measurements. The optical forces of laser tweezers enable precise (subnanometer) localization of the trapped object, and permit application of precisely calibrated point forces for the pur...

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confidence: 99%

Electrokinetic trapping at the one nanometer limit

Fields

Cohen

2011

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

Self Cite

133

155

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“…In the latter work, camera images are replaced by a continuously scanning laser and a single-pixel detector that detects individual photon counts. 3,27 Each individual count leads to an updated estimate of particle position, using a modified Kalman filter. The advantage of such a setup is vastly increased speed and a simplicity that comes from having instantaneous estimates of particle position (no camera exposure effects).…”

Section: Discussionmentioning

confidence: 99%

Real-Time Calibration of a Feedback Trap

Gavrilov¹

2017

Experiments on the Thermodynamics of Information Processing

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Feedback traps use closed-loop control to trap or manipulate small particles and molecules in solution. They have been applied to the measurement of physical and chemical properties of particles and to explore fundamental questions in the non-equilibrium statistical mechanics of small systems. These applications have been hampered by drifts in the electric forces used to manipulate the particles. Although the drifts are small for measurements on the order of seconds, they dominate on time scales of minutes or slower. Here, we show that an extended recursive least-squares (RLS) parameter-estimation algorithm can allow real-time measurement and control of electric and stochastic forces over time scales of hours. Simulations show that the extended-RLS algorithm recovers known parameters accurately. Experimental estimates of diffusion coefficients are also consistent with expected physical properties.

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“…[100][101][102][103][104][105] Briefly, the device consists of a crosslike channel arrangement with a smallest width of about 20 mm and a height of about 1 mm. Figure 4 shows a two-dimensional crosssection with the main components of the ABELtrap.…”

Section: Breaking Diffusion Limitationsmentioning

confidence: 99%

Improving FRET‐Based Monitoring of Single Chemomechanical Rotary Motors at Work

Börsch

Wrachtrup

2011

ChemPhysChem

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Protein dynamics are observable in real time using internal distance measurements of reference positions. Based on Förster resonance energy transfer (FRET) as the distance ruler between two fluorescent markers, investigations of single molecules one at a time have modified our views of protein conformational changes. X-ray crystallography provides trapped conformations at atomic resolution and NMR can add information about flexible parts of proteins, but the pathways between these structures including unknown conformations are often not directly accessible. Especially for membrane enzymes comprising multiple subunits, limited structural information requires alternative experimental methods to follow conformational pathways. Herein we summarize the knowledge gathered from single-molecule FRET studies of the membrane-embedded rotary nanomotor F(o)F(1)-ATP synthase. In addition, new ideas and concepts to shift and extend the current limitations of the confocal FRET detection approach using freely diffusing, liposome-reconstituted, individual enzymes are discussed: nanodiamonds as new fluorophores, optimized laser excitation schemes, Hidden Markov Models for software-based FRET trajectory analysis, and application of the anti-Brownian electrokinetic trap (ABELtrap) to keep the single enzyme in focus.

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Hardware-based anti-Brownian electrokinetic trap (ABEL trap) for single molecules: control loop simulations and application to ATP binding stoichiometry in multi-subunit enzymes

Cited by 23 publications

References 26 publications

Electrokinetic trapping at the one nanometer limit

Electrokinetic trapping at the one nanometer limit

Real-Time Calibration of a Feedback Trap

Improving FRET‐Based Monitoring of Single Chemomechanical Rotary Motors at Work

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