Virginia VanDelinder

Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing.

We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution.

Click strategies for single-molecule protein fluorescence.

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Microfluidic Device for Single-Molecule Experiments with Enhanced Photostability

A microfluidic device made of polydimethylsiloxane (PDMS) addresses key limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Photobleaching is dramatically reduced by deoxygenation via gas diffusion through porous channel walls. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation minimizes surface-sample contacts and allows the in situ addition and combination of other reagents.

Continuous throughput and long-term observation of single-molecule FRET without immobilization

We present an automated microfluidic platform that performs multisecond observation of single molecules with millisecond time resolution while bypassing the need for immobilization procedures. With this system, we confine biomolecules to a thin excitation field by reversibly collapsing microchannels to nanochannels. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA Holliday junctions, nucleosomes and human transglutaminase 2.

Bioluminescent response of individual dinoflagellate cells to hydrodynamic stress measured with millisecond resolution in a microfluidic device

SUMMARY Dinoflagellate bioluminescence serves as a model system for examining mechanosensing by suspended motile unicellular organisms. The response latency, i.e. the delay time between the mechanical stimulus and luminescent response, provides information about the mechanotransduction and signaling process, and must be accurately known for dinoflagellate bioluminescence to be used as a flow visualization tool. This study used a novel microfluidic device to measure the response latency of a large number of individual dinoflagellates with a resolution of a few milliseconds. Suspended cells of several dinoflagellate species approximately 35 μm in diameter were directed through a 200 μm deep channel to a barrier with a 15 μm clearance impassable to the cells. Bioluminescence was stimulated when cells encountered the barrier and experienced an abrupt increase in hydrodynamic drag, and was imaged using high numerical aperture optics and a high-speed low-light video system. The average response latency for Lingulodinium polyedrum strain HJ was 15 ms (N>300 cells) at the three highest flow rates tested, with a minimum latency of 12 ms. Cells produced multiple flashes with an interval as short as 5 ms between individual flashes, suggesting that repeat stimulation involved a subset of the entire intracellular signaling pathway. The mean response latency for the dinoflagellates Pyrodinium bahamense, Alexandrium monilatum and older and newer isolates of L. polyedrum ranged from 15 to 22 ms, similar to the latencies previously determined for larger dinoflagellates with different morphologies, possibly reflecting optimization of dinoflagellate bioluminescence as a rapid anti-predation behavior.

High-Resolution Temperature−Concentration Diagram of α-Synuclein Conformation Obtained from a Single Förster Resonance Energy Transfer Image in a Microfluidic Device

We present a microfluidic device for rapid and efficient determination of protein conformations in a range of medium conditions and temperatures. The device generates orthogonal gradients of concentration and temperature in an interrogation area that fits into the field of view of an objective lens with a numerical aperture of 0.45. A single Förster resonance energy transfer (FRET) image of the interrogation area containing a dual-labeled protein provides a 100 x 100 point map of the FRET efficiency that corresponds to a diagram of protein conformations in the coordinates of temperature and medium conditions. The device is used to explore the conformations of alpha-synuclein, an intrinsically disordered protein linked to Parkinsons and Alzheimers diseases, in the presence of a binding partner, the lipid-mimetic sodium dodecyl sulfate (SDS). The experiment provides a diagram of conformations of alpha-synuclein with 10,000 individual data points in a range of 21-47 degrees C and 0-2.5 mM SDS. The diagram is consistent with previous reports but also reveals new conformational transitions that would be difficult to detect with conventional techniques. The microfluidic device can potentially be used to study other biomolecular and soft-matter systems.

Photodamage and the Importance of Photoprotection in Biomolecular-Powered Device Applications

In recent years, an enhanced understanding of the mechanisms underlying photobleaching and photoblinking of fluorescent dyes has led to improved photoprotection strategies, such as reducing and oxidizing systems (ROXS) that reduce blinking and oxygen scavenging systems to reduce bleaching. Excitation of fluorescent dyes can also result in damage to catalytic proteins (e.g., biomolecular motors), affecting the performance of integrated devices. Here, we characterized the motility of microtubules driven by kinesin motor proteins using various photoprotection strategies, including a microfluidic deoxygenation device. Impaired motility of microtubules was observed at high excitation intensities in the absence of photoprotection as well as in the presence of an enzymatic oxygen scavenging system. In contrast, using a polydimethylsiloxane (PDMS) microfluidic deoxygenation device and ROXS, not only were the fluorophores slower to bleach but also moving the velocity and fraction of microtubules over time remained unaffected even at high excitation intensities. Further, we demonstrate the importance of photoprotection by examining the effect of photodamage on the behavior of a switchable mutant of kinesin. Overall, these results demonstrate that improved photoprotection strategies may have a profound impact on functional fluorescently labeled biomolecules in integrated devices.

The role of membrane fluidization in the gel-assisted formation of giant polymersomes

Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.

Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.

Mechanisms Underlying the Active Self-Assembly of Microtubule Rings and Spools.

Active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly. Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications.