Single-molecule FRET combined with electrokinetic trapping reveals real-time enzyme kinetics of individual F-ATP synthases.

Single-molecule Förster resonance energy transfer (smFRET) is a key technique to observe conformational changes in molecular motors and to access the details of single-molecule static and dynamic disorder during catalytic processes. However, studying freely diffusing molecules in solution is limited to a few tens of milliseconds, while surface attachment often bears the risk to restrict their natural motion. In this paper we combine smFRET and electrokinetic trapping (ABEL trap) to non-invasively hold single FOF1-ATP synthases for up to 3 s within the detection volume, thereby extending the observation time by a factor of 10 as compared to Brownian diffusion without surface attachment. In addition, we are able to monitor complete reaction cycles and to selectively trap active molecules based on their smFRET signal, thus speeding up the data acquisition process. We demonstrate the capability of our method to study the dynamics of single molecules by recording the ATP-hydrolysis driven rotation of individual FOF1-ATP synthase molecules over numerous reaction cycles and extract their kinetic rates. We argue that our method is not limited to motor proteins. Instead, it can be applied to monitor conformational changes with millisecond time resolution for a wide range of enzymes, thereby making it a versatile tool for studying protein dynamics.

Single-molecule FRET dynamics of molecular motors in an ABEL trap.

Single-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap's inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. In this work we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 seconds with sub-millisecond time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

Probing Ligand-Receptor Interaction in Living Cells Using Force Measurements With Optical Tweezers.

This work probes the binding kinetics of COOH-terminus of Clostridium perfringens enterotoxin (c-CPE) and claudin expressing MCF-7 cells using force spectroscopy with optical tweezers. c-CPE is of high biomedical interest due to its ability to specifically bind to claudin with high affinity as well as reversibly disrupt tight junctions whilst maintaining cell viability. We observed single-step rupture events between silica particles functionalized with c-CPE and MCF-7 cells. Extensive calibration of the optical tweezers' trap stiffness and displacement of the particle from trap center extracted a probable bond rupture force of ≈ 18 pN. The probability of rupture events with c-CPE functionalized silica particles increased by 50% compared to unfunctionalized particles. Additionally, rupture events were not observed when probing cells not expressing claudin with c-CPE coated particles. Overall, this work demonstrates that optical tweezers are invaluable tools to probe ligand-receptor interactions and their potential to study dynamic molecular events in drug-binding scenarios.

Optically trapped bacteria pairs reveal discrete motile response to control aggregation upon cell-cell approach.

Aggregation of bacteria plays a key role in the formation of many biofilms. The critical first step is cell-cell approach, and yet the ability of bacteria to control the likelihood of aggregation during this primary phase is unknown. Here, we use optical tweezers to measure the force between isolated Bacillus subtilis cells during approach. As we move the bacteria towards each other, cell motility (bacterial swimming) initiates the generation of repulsive forces at bacterial separations of ~3 µm. Moreover, the motile response displays spatial sensitivity with greater cell-cell repulsion evident as inter-bacterial distances decrease. To examine the environmental influence on the inter-bacterial forces, we perform the experiment with bacteria suspended in Tryptic Soy Broth, NaCl solution and deionised water. Our experiments demonstrate that repulsive forces are strongest in systems that inhibit biofilm formation (Tryptic Soy Broth), while attractive forces are weak and rare, even in systems where biofilms develop (NaCl solution). These results reveal that bacteria are able to control the likelihood of aggregation during the approach phase through a discretely modulated motile response. Clearly, the force-generating motility we observe during approach promotes biofilm prevention, rather than biofilm formation.

Measuring nanoparticle flow with the image structure function.

We present a technique to measure the velocity and flow profiles of a nanofluid in a microfluidic channel. Importantly, we extract the flow velocity from a series of standard brightfield images without employing particle tracking or laser-enhanced methods. Our analysis retrieves the flow information from the image structure function of sub-diffraction limited nanoparticles in suspension. We are able to spatially resolve the flow velocity and map out the parabolic flow profile across the width of a microfluidic channel.

Holographic aberration correction: optimising the stiffness of an optical trap deep in the sample.

We investigate the effects of 1(st) order spherical aberration and defocus upon the stiffness of an optical trap tens of µm into the sample. We control both these aberrations with a spatial light modulator. The key to maintain optimum trap stiffness over a range of depths is a specific non-trivial combination of defocus and axial objective position. This optimisation increases the trap stiffness by up to a factor of 3 and allows trapping of 1 µm polystyrene beads up to 50 µm deep in the sample.

Optical vortex trap for resonant confinement of metal nanoparticles.

The confinement and controlled movement of metal nanoparticles and nanorods is an emergent area within optical micromanipulation. In this letter we experimentally realise a novel trapping geometry near the plasmon resonance using an annular light field possessing a helical phasefront that confines the nanoparticle to the vortex core (dark) region. We interpret our data with a theoretical framework based upon the Maxwell stress tensor formulation to elucidate the total forces upon nanometric particles near the particle plasmon resonance. Rotation of the particle due to orbital angular momentum transfer is observed. This geometry may have several advantages for advanced manipulation of metal nanoparticles.