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.

ReuseUnless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. Real-time dynamic substructuring is a novel experimental technique used to test the dynamic behaviour of complex structures. The technique involves creating a hybrid model of the entire structure by combining an experimental test piece -the substructure -with a set of numerical models. In this paper we describe a multi-actuator substructured system of a coupled three mass-spring-damper system and use this to demonstrate the nature of delay errors which can first lead to a loss of accuracy and then to instability of the substructuring algorithm.Synchronisation theory and delay compensation are used to show how the delay errors, present in the transfer systems, can be minimised by online forward prediction. This new algorithm uses a more generic approach than the single step algorithms applied to substructuring thus far, giving considerable advantages in terms of flexibility and accuracy. The basic algorithm is then extended by closing the control loop resulting in an error driven adaptive feedback controller which can operate with no prior knowledge of the plant dynamics. The adaptive algorithm is then used to perform a real substructuring test using experimentally measured forces to deliver a stable substructuring algorithm.

The motions of a dye-labeled DNA hairpin loop (Cy5-5′-GGGTT-(A) 30 -AACCC-3′-TMR) have been investigated through the fluctuations in proximity ratio from fluorescence resonance energy transfer (FRET). We examine three solution conditions: (1) MilliQ water, (2) Tris-EDTA buffer, and (3) Tris-EDTA buffer plus an excess of DNA complementary to the loop sequence, (T) 30 . Correlations in proximity ratio show submillisecond dynamics. Static heterogeneity is revealed from the distribution of proximity ratio amplitudes. The observed stretched exponential kinetics are consistent with a model based on the transition between two states over a complex energy landscape.

SUMMARYReal-time dynamic substructuring is an experimental technique for testing the dynamic behaviour of complex structures. It involves creating a hybrid model of the entire structure by combining an experimental test piece-the substructure-with a numerical model describing the remainder of the system. The technique is useful when it is impractical to experimentally test the entire structure or complete numerical modelling is insu cient.In this paper, we focus on the in uence of delay in the system, which is generally due to the inherent dynamics of the transfer systems (actuators) used for structural testing. This naturally gives rise to a delay di erential equation (DDE) model of the substructured system. With the case of a substructured system consisting of a single mass-spring oscillator we demonstrate how a DDE model can be used to understand the in uence of the response delay of the actuator. Speciÿcally, we describe a number of methods for identifying the critical time delay above which the system becomes unstable.Because of the low damping in many large structures a typical situation is that a substructuring test would operate in an unstable region if additional techniques were not implemented in practice. We demonstrate with an adaptive delay compensation technique that the substructured mass-spring oscillator system can be stabilized successfully in an experiment. The approach of DDE modelling also allows us to determine the dependence of the critical delay on the parameters of the delay compensation scheme. Using this approach we develop an over-compensation scheme that will help ensure stable experimental testing from initiation to steady state operation. This technique is particularly suited to sti structures or those with very low natural damping as regularly encountered in structural engineering.

We describe a protocol for forming an artificial lipid bilayer by contacting nanoliter aqueous droplets in an oil solution in the presence of phospholipids. A lipid monolayer forms at each oil-water interface, and when two such monolayers touch, a bilayer is created. Droplet interface bilayers (DIBs) are a simple way to generate stable bilayers suitable for single-channel electrophysiology and optical imaging from a wide variety of preparations, ranging from purified proteins to reconstituted eukaryotic cell membrane fragments. Examples include purified proteins from the α-hemolysin pore from Staphylococcus aureus, the anthrax toxin pore and the 1.2-MDa mouse mechanosensitive channel MmPiezo1. Ion channels and ionotropic receptors can also be reconstituted from membrane fragments without further purification. We describe two approaches for forming DIBs. In one approach, a lipid bilayer is created between two aqueous droplets submerged in oil. In the other approach, a membrane is formed between an aqueous droplet and an agarose hydrogel, which allows imaging in addition to electrical recordings. The protocol takes <30 min, including droplet generation, monolayer assembly and bilayer formation. In addition to the main protocol, we also describe the preparation of Ag/AgCl electrodes and sample preparation.