A microreactor sealing method using adhesive tape for digital bioassays.

Digital assays using microreactors fabricated on solid substrates are useful for carrying out sensitive assays of infectious diseases and other biological tests. However, sealing of the microchambers using fluid oil is difficult for non-experts, and thus hinders the widespread use of digital microreactor assays. Here, we propose the physical isolation of tiny reactors with adhesive tape (PITAT) using simple, commercially available pressure-sensitive adhesive (PSA) tape as a separator of the microreactors. We confirmed that PSA tape can effectively seal the microreactors and prevent molecules from diffusing out. By testing several types of adhesive tape, we found that rubber-based adhesives are the most suitable for this purpose. In addition, we demonstrated that single-molecule enzyme assays can be successfully performed inside microreactors sealed with PSA tape. The results obtained using PITAT are quantitatively comparable to conventional oil sealing, although it is quick and cost-effective. Finally, we demonstrated that single-particle virus counting of the influenza virus can be achieved using PITAT. Collectively, our results suggest that PITAT may be suitable for use in the design of sensitive tests for infectious diseases at the point of care, where no sophisticated equipment or machines are available.

Osmolyte-Enhanced Protein Synthesis Activity of a Reconstituted Translation System.

Molecular crowding is receiving great attention in cell-free synthetic biology because molecular crowding is a critical feature of natural cell discrimination from artificial cells. Further, it has significant and generic influences on biomolecular functions. Although there are reports on how the macromolecular crowder reagents affect cell-free systems such as transcription and translation, the second class of molecular crowder reagents with low molecular weight, osmolyte, was much less studied in cell-free systems. In the present study, we focused on trimethylamine- N-oxide (TMAO) and betaine, methylamine osmolytes, and investigated the effectiveness of these osmolytes on gene expression activity of reconstituted cell-free protein synthesis. The gene expression activity of the fluorescent proteins Venus and tdTomato and the enzymes ß-galactosidase and dihydrofolate reductase were tested. At 37 °C, 0.4 M TMAO showed the highest enhancement of translational activity by a factor of 1.6-3.8, regardless of protein type. In contrast, betaine showed only a moderate effect that was limited to fluorescent proteins. Excess amounts of osmolytes suppressed gene expression activity. An mRNA-start assay and SDS-PAGE quantitative analysis provided firm evidence that TMAO enhances the translation process, instead of transcription, folding, or the maturation of fluorescent proteins. Interestingly, at 26 °C, TMAO and betaine showed the highest enhancement of protein synthesis activity at lower concentrations than at 37 °C. These findings provide implications on how osmolytes assist translation in natural cells. Further, they provide guidelines for modulation of protein synthesis activity in artificial cells through osmolyte addition.

Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging.

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator "QUEEN", which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells.

Grafting synthetic transmembrane units to the engineered low-toxicity α-hemolysin to restore its hemolytic activity.

The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.e., the replacement of a functional domain of proteins with alternative chemical compounds to build "cyborg proteins." As a proof of concept model, we chose staphylococcal α-hemolysin (Hla), which is a well-studied, pore-forming toxin. The hemolytic activity of Hla mutants was dramatically decreased by truncation of the stem domain, which forms a ß-barrel pore in the membrane. However, the impaired hemolytic activity was significantly restored by attaching a pyrenyl-maleimide unit to the cysteine residue that was introduced in the remaining stem domain. In contrast, negatively charged fluorescein-maleimide completely abolished the remaining activity of the mutants.

Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity.

Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-µm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins' function.

Robustness of the rotary catalysis mechanism of F1-ATPase.

F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought.

Biased Brownian stepping rotation of FoF1-ATP synthase driven by proton motive force.

FoF1-ATP synthase (FoF1) produces most of the ATP in cells, uniquely, by converting the proton motive force (pmf) into ATP production via mechanical rotation of the inner rotor complex. Technical difficulties have hampered direct investigation of pmf-driven rotation, which are crucial to elucidating the chemomechanical coupling mechanism of FoF1. Here we develop a novel supported membrane system for direct observation of the rotation of FoF1 driven by pmf that was formed by photolysis of caged protons. Upon photolysis, FoF1 initiated rotation in the opposite direction to that of the ATP-driven rotation. The step size of pmf-driven rotation was 120°, suggesting that the kinetic bottleneck is a catalytic event on F1 with threefold symmetry. The reaction equilibrium was slightly biased to ATP synthesis like under physiological conditions, and FoF1 showed highly stochastic behaviour, frequently making a 120° backward step. This new experimental system would be applicable to single-molecule study of other membrane proteins.

Role of the DELSEED loop in torque transmission of F1-ATPase.

F(1)-ATPase is an ATP-driven rotary motor that generates torque at the interface between the catalytic ß-subunits and the rotor γ-subunit. The ß-subunit inwardly rotates the C-terminal domain upon nucleotide binding/dissociation; hence, the region of the C-terminal domain that is in direct contact with γ-termed the DELSEED loop-is thought to play a critical role in torque transmission. We substituted all the DELSEED loop residues with alanine to diminish specific DELSEED loop-γ interactions and with glycine to disrupt the loop structure. All the mutants rotated unidirectionally with kinetic parameters comparable to those of the wild-type F(1), suggesting that the specific interactions between DELSEED loop and γ is not involved in cooperative interplays between the catalytic ß-subunits. Glycine substitution mutants generated half the torque of the wild-type F(1), whereas the alanine mutant generated comparable torque. Fluctuation analyses of the glycine/alanine mutants revealed that the γ-subunit was less tightly held in the α(3)ß(3)-stator ring of the glycine mutant than in the wild-type F(1) and the alanine mutant. Molecular dynamics simulation showed that the DELSEED loop was disordered by the glycine substitution, whereas it formed an α-helix in the alanine mutant. Our results emphasize the importance of loop rigidity for efficient torque transmissions.

Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution.

Molecular motors such as kinesin, myosin, and F(1)-ATPase are responsible for many important cellular processes. These motor proteins exhibit nanometer-scale, stepwise movements on micro- to millisecond timescales. So far, methods developed to measure these small and fast movements with high spatial and temporal resolution require relatively complicated experimental systems. Here, we describe a simple dark-field imaging system that employs objective-type evanescent illumination to selectively illuminate a thin layer on the coverslip and thus yield images with high signal/noise ratios. Only by substituting the dichroic mirror in conventional objective-type total internal reflection fluorescence microscope with a perforated mirror, were nanometer spatial precision and microsecond temporal resolution simultaneously achieved. This system was applied to the study of the rotary mechanism of F(1)-ATPase. The fluctuation of a gold nanoparticle attached to the gamma-subunit during catalytic dwell and the stepping motion during torque generation were successfully visualized with 9.1-mus temporal resolution. Because of the simple optics, this system will be applicable to various biophysical studies requiring high spatial and temporal resolution in vitro and also in vivo.

Sequential processing from cell lysis to protein assay on a chip enabling the optimization of an F(1)-ATPase single molecule assay condition.

We developed an integrated protein assay device, "Single Molecule MicroTAS (SMM)," which enables cell lysis, protein extraction, purification, and activity assay. The assay was achieved at the single-molecule scale for a genetically engineered protein, F(1)-ATPase, which is the smallest known rotary motor. A cell lysis condition, with a wide range of applied voltages (50-250 V) and other optimized values (pulse width: 50 micros; duty: 0.01%; electrode gap: 25 microm; total flow rate: 5 microL min(-1)) provided a high enough protein concentration for the assay. Successively, the protein was extracted and purified by specific binding in a microfluidic channel. During the assay process, the diffusion effect of lysate between a two-phase laminar flow contributes to optimizing the single-molecule assay condition, because the concentration of the original lysate from the E. coli solution is too high to assay. To achieve the most efficient assay condition, the protein diffusion effect on the assay was experimentally and numerically evaluated. The results reveal that, in our experimental conditions, concentrations of F(1) and other contaminated effluents are optimized for the F(1) rotational assay at a channel position. The adenosine triphosphate (ATP)-driven rotation speed measured in the SMM was compatible with that obtained by conventional purification and assay. Such a sequential process from cell lysis to assay proves that the SMM is an example of a sample-in-answer-out system for F(1) protein evaluation.

Acceleration of the ATP-binding rate of F1-ATPase by forcible forward rotation.

F1-ATPase (F1) is a reversible ATP-driven rotary motor protein. When its rotary shaft is reversely rotated, F1 produces ATP against the chemical potential of ATP hydrolysis, suggesting that F1 modulates the rate constants and equilibriums of catalytic reaction steps depending on the rotary angle of the shaft. Although the chemomechanical coupling scheme of F1 has been determined, it is unclear how individual catalytic reaction steps depend on its rotary angle. Here, we report direct evidence that the ATP-binding rate of F1 increases upon the forward rotation of the rotor, and its binding affinity to ATP is enhanced by rotation.

Mechanism of inhibition by C-terminal alpha-helices of the epsilon subunit of Escherichia coli FoF1-ATP synthase.

The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

Photo gel-sol/sol-gel transition and its patterning of a supramolecular hydrogel as stimuli-responsive biomaterials.

In a focused library of glycolipid-based hydrogelators bearing fumaric amide as a trans-cis photoswitching module, several new photoresponsive supramolecular hydrogelators were discovered, the gel-sol/sol-gel transition of which was pseudo-reversibly induced by light. Studying the optimal hydrogel by NMR spectroscopy and various microscopy techniques showed that the trans-cis photoisomerization of the double bond of the fumaric amide unit effectively caused assembly or disassembly of the self-assembled supramolecular fibers to yield the macroscopic hydrogel or the corresponding sol, respectively. The entanglement of the supramolecular fibers produced nanomeshes, the void space of which was roughly evaluated to be 250 nm based on confocal laser scanning microscopy observations of the size-dependent Brownian motion of nanobeads embedded in the supramolecular hydrogel. It was clearly shown that such nanomeshes become a physical obstacle that captures submicro- to micrometer-sized substrates such as beads or bacteria. By exploiting the photoresponsive property of the supramolecular nanomeshes, we succeeded in off/on switching of bacterial movement and rotary motion of bead-tethered F(1)-ATPase, a biomolecular motor protein, in the supramolecular hydrogel. Furthermore, by using the photolithographic technique, gel-sol photopatterning was successfully conducted to produce sol spots within the gel matrix. The fabricated gel-sol pattern not only allowed regulation of bacterial motility in a limited area, but also off/on switching of F1-ATPase rotary motion at the single-molecule level. These results demonstrated that the photoresponsive supramolecular hydrogel and the resulting nanomeshes may provide unique biomaterials for the spatiotemporal manipulation of various biomolecules and live bacteria.

Thermally responsive supramolecular nanomeshes for on/off switching of the rotary motion of F1-ATPase at the single-molecule level.

The artificial regulation of protein functions is essential for the realization of protein-based soft devices, because of their unique functions conducted within a nano-sized molecular space. We report that self-assembled nanomeshes comprising heat-responsive supramolecular hydrogel fibers can control the rotary motion of an enzyme-based biomotor (F(1)-ATPase) in an on/off manner at the single-molecule level. Direct observation of the interaction of the supramolecular fibers with a microbead unit tethered to the F(1)-ATPase and the clear threshold in the size of the bead required to stop ATPase rotation indicates that the bead was physically blocked so as to stop the rotary motion of ATPase. The temperature-induced formation and collapse of the supramolecular nanomesh can produce or destroy, respectively, the physical obstacle for ATPase so as to control the ATPase motion in an off/on manner. Furthermore, this switching of the F(1)-ATPase motion could be spatially restricted by using a microheating device. The integration of biomolecules and hard materials, interfaced with intelligent soft materials such as supramolecular hydrogels, is promising for the development of novel semi-synthetic nano-biodevices.