Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

DNA hairpin-loop structures fluctuate between different conformations and are generally classified as open or closed (as shown in Fig. 1). They are involved in various biological functions, including gene expression and regulation (5, 6), and more recently they have found use as DNA biosensors (e.g., molecular beacons) (7,8).The open-to-closed transition provides a simple case with which to study the dynamics of intramolecular chain diffusion. Continuing this theme, we developed a method for detecting biopolymer conformational dynamics based on the fluctuations in proximity ratio from FRET (10). By attaching donor and acceptor fluorophores to both ends of a DNA hairpin, the open-to-closed conformational dynamics of the system can be detected at ultra-high sensitivity down to the single-molecule level. By constructing the autocorrelation function of the proximity ratio rather than of the fluorescence intensity, we simplify the extraction of intramolecular kinetics from the correlation function. The use of the ratiometric method should result in a correlation function independent of molecular diffusion (10). This new approach enables us to observe stretched exponential kinetics for the conformational fluctuation in a DNA hairpinloop system (10). A multiple-pathway, two-state model was proposed and used to simulate experimental single-molecule proximity ratio distributions (11).In our previous studies, we concentrated on the time scale of the observed fluctuation and did not fully exploit the amplitude of the autocorrelation function. Here, we report the investigation of temperature and viscosity dependence for the conformational fluctuation of a DNA hairpin-loop. We show that the correlation amplitude is directly related to the equilibrium constant of the open-to-closed transition. By using a two-state model we are able to recover the apparent thermodynamic and kinetic parameters for the hairpin-loop conformational fluctuations under different buffer conditions. The kinetics for the open-to-closed transition of the hairpin-loop appear to show non-Arrhenius behavior akin to that found in peptide ␤-hairpins (12). Materials and MethodsA 40-base oligonucleotide 5Ј-GGGTT-(A) 30 -AACCC-3Ј was chosen as our model DNA hairpin-loop. Donor fluorophore carboxytetramethylrhodamine (TMR) is attached at its 3Ј end via a modified cytosine and a six-carbon linker. Acceptor fluorophore indodicarbocyanine (Cy5) is attached at its 5Ј end via a three-carbon linker. The donor and acceptor form a fluorescence resonance energy transfer pair with a FRET distance (R 0 ) of Ϸ5.3 nm. Both dual-labeled and singly (TMR) labeled oligonucleotides were purchased from Operon Technologies (Alameda, CA) and were HPLC purified. The structure of the fully closed hairpin-loop is illustrated in Fig. 1.The viscosity was varied by adding up to 55% (in mass) of glycerol (molecular biology reagent from Sigma) to the aqueous solution. The precise viscosity of the mixture was calculated by using a polynomial fit to the tabulated viscosity of water͞glycerol m...

By making dynamic changes to the area of a droplet interface bilayer (DIB), we are able to measure the specific capacitance of lipid bilayers with improved accuracy and precision over existing methods. The dependence of membrane specific capacitance on the chain-length of the alkane oil present in the bilayer is similar to that observed in black lipid membranes. In contrast to conventional artificial bilayers, DIBs are not confined by an aperture, which enables us to determine that the dependence of whole bilayer capacitance on applied potential is predominantly a result of a spontaneous increase in bilayer area. This area change arises from the creation of new bilayer at the three phase interface and is driven by changes in surface tension with applied potential that can be described by the Young-Lippmann equation. By accounting for this area change, we are able to determine the proportion of the capacitance dependence that arises from a change in specific capacitance with applied potential. This method provides a new tool with which to investigate the vertical compression of the bilayer and understand the changes in bilayer thickness with applied potential. We find that, for 1,2-diphytanoyl-sn-glycero-3-phosphocholine membranes in hexadecane, specific bilayer capacitance varies by 0.6-1.5% over an applied potential of ±100 mV.

The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.