Compartments for the spatially and temporally controlled assembly of biological processes are essential towards cellular life. Synthetic mimics of cellular compartments based on lipid-based protocells lack the mechanical and chemical stability to allow their manipulation into a complex and fully functional synthetic cell. Here, we present a high-throughput microfluidic method to generate stable, defined sized liposomes termed 'droplet-stabilized giant unilamellar vesicles (dsGUVs)'. The enhanced stability of dsGUVs enables the sequential loading of these compartments with biomolecules, namely purified transmembrane and cytoskeleton proteins by microfluidic pico-injection technology. This constitutes an experimental demonstration of a successful bottom-up assembly of a compartment with contents that would not self-assemble to full functionality when simply mixed together. Following assembly, the stabilizing oil phase and droplet shells are removed to release functional self-supporting protocells to an aqueous phase, enabling them to interact with physiologically relevant matrices.

This article reports stable photoluminescence and high-contrast optically detected electron spin resonance (ODESR) from single nitrogen-vacancy (NV) defect centers created within ultrasmall, disperse nanodiamonds of radius less than 4 nm. Unexpectedly, the efficiency for the production of NV fluorescent defects by electron irradiation is found to be independent of the size of the nanocrystals. Fluorescence lifetime imaging shows lifetimes with a mean value of around 17 ns, only slightly longer than the bulk value of the defects. After proper surface cleaning, the dephasing times of the electron spin resonance in the nanocrystals approach values of some microseconds, which is typical for the type Ib diamond from which the nanoparticle is made. We conclude that despite the tiny size of these nanodiamonds the photoactive nitrogen-vacancy color centers retain their bulk properties to the benefit of numerous exciting potential applications in photonics, biomedical labeling, and imaging.

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

F 0 F 1 -ATP synthases catalyze proton transport-coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the e-subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescencelabeled F 0 F 1 from Escherichia coli was incorporated into liposomes. Single-molecule fluorescence resonance energy transfer (FRET) revealed that the e-subunit rotates stepwise showing three distinct distances to the b-subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of e during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of e. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active-inactive transition was associated with a conformational change of e within the central stalk.

Synthesis of adenosine triphosphate ATP, the 'biological energy currency', is accomplished by F(o)F(1)-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the F(o) motor powers catalysis in the F(1) motor. Although F(1) uses 120 degrees stepping during ATP synthesis, models of F(o) predict either an incremental rotation of c subunits in 36 degrees steps or larger step sizes comprising several fast substeps. Using single-molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36 degrees sequential stepping mode of the c-ring during ATP synthesis.

The EF 0 F 1 -ATP synthase mutants bQ64C and Q QT106C were labelled selectively with the £uorophores tetramethylrhodamine (TMR) at the b-subunit and with a cyanine (Cy5) at the Q Q-subunit. After reconstitution into liposomes, these double-labelled enzymes catalyzed ATP synthesis at a rate of 33 s 31 . Fluorescence of TMR and Cy5 was measured with a confocal set-up for single-molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with di¡erent £uorescence resonance energy transfer (FRET) e⁄ciencies were observed. In the presence of ATP, repeating sequences of those three FRETstates were identi¢ed, indicating stepwise rotation of the Q Q-subunit of EF 0 F 1 . ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Transforming micelle structure: Amphiphilic DNA block copolymers form spherical micelles in solution. They can be transformed into rodlike micelles by hybridization with long DNA sequences, which consist of parallel aligned double‐stranded DNA molecules “glued” together by hydrophobic interactions of the organic polymer (see picture). The template determines the length of the rodlike aggregates.

Self-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors' active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature's nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme's diffusion due to active self-propulsion must be observed on top of the enzyme's passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme's substrate (and sometimes its inhibitor) of up to 80% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme's diffusion. We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated. We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes' catalytic activity to their self-propulsion. We hope that our findings hel...