Eric C. Freeman

Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.

MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1–3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4–6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

Deterministic model of biomolecular networks with stimuli-responsive properties

A deterministic model is presented for predicting the passive and stimuli-responsive characteristics of biomolecular networks. These biomolecular networks consist of multiple bilayer interfaces that can incorporate stimuli-responsive molecules such as peptides and proteins. A model is presented and a lumped parameter method is used to obtain the network equations for several case studies. The model is then utilized to predict the response of a biomolecular network that exhibits rectification behavior due to the presence of voltage-responsive protein incorporated into the interfacial bilayer. This article demonstrates the electric response of systems of biomolecular networks and prompts further research into their use for engineering applications.

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers.

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Mechanosensitive droplet interface bilayer networks

A method for studying the coupled electrical-mechanical response of droplet interface bilayers is proposed. This research examines the concept of the biologically-inspired hair cell in greater depth, attempting to determine the source of the sensing current when no external potential is applied across the sensing droplet-interface bilayer element. Historically the mechanosensitive current in these droplet-interface bilayers has been attributed to a combination of capacitive currents and electrode oscillation (experimental error); however the development of a third sensing mechanism through modifying the bilayer properties may enhance the usefulness of the mechanosensitive droplet interface bilayer networks considerably. This would allow for measurable sensing currents without requiring an externally applied electric field by permanently charging the bilayer element through surface modifications. Charging agents are added to the droplet interface bilayer network as the network is oscillated and the electrical response is recorded for analysis. The adsorption of the charged molecules is studied through the intramembrane field compensation (IFC) approach, and the knowledge gained from this is then applied towards the mechanosensitivity analysis. Multiple charging techniques are tested and employed, and the nature of the sensing current is determined by examining the frequency content of the recorded currents. Several properties are derived, including the nature of the sensing current, the charging mechanisms available for boosting the sensing current, and the nature of the sensing current without externally applied potentials.

Using cellular energy conversion and storage mechanics for bio-inspired energy harvesting

Novel biologically-inspired energy harvesting devices constructed with lipid bilayer membranes are studied. Recently the research group has proposed the use of biomolecular unit cells consisting of encapsulated droplets with a lipid bilayer formed at their interfaces, stabilized between the two aqueous compartments. This allows for the rapid study and assessment of the characteristics of the individual unit cell, the insertion of various transport proteins and peptides that shape the response of the unit cell, and the construction of complex networks of these biomolecular systems. The goal of this work is to develop and study methods for constructing energy relevant devices through these biomolecular networks. These networks are highly tailorable, and allow the researcher to alter the embedded proteins/peptides in the lipid bilayer, the bilayer dimensions through the application of compressive forces, and the salt concentrations in the droplets. This allows for a high degree of control over their attributes and outputs. These systems also exhibit collective properties through large networks of the unit cells, allowing for complex sensing and actuation behavior not exhibited by single cells. This paper provides an overview of the development of a model for predicting the performance and output of these energy relevant biomolecular networks as well as preliminary experimental results that demonstrate some of the concepts in action.

The influence of osmotic pressure on the lifespan of cellularly inspired energy-relevant materials

Bimolecular unit cells have recently become a focus for biologically-inspired smart materials. This is largely due their ability to exhibit many of the same properties as the natural cell membrane. In this study, two lipid monolayers formed at a water/oil interface are brought together, creating a lipid bilayer at their interface with each droplet containing a different concentration of ions. This ionic concentration gradient leads to the development of a membrane potential across the bilayer as ions begin to passively diffuse across the membrane at varying rates, providing the proof of concept for energy storage through cellular mechanics. The focus of the study is to determine the influence of osmotic pressure on the lifespan of the lipid bilayer. We hypothesize that the greater osmotic pressure that develops from a greater ionic concentration gradient will prove to have a negative impact on the lifespan of the bilayer membrane, causing it to rupture sooner. This is due to the substantial amount of osmotic swelling that will occur to compensate for the ionic concentration gradient. This study will demonstrate how osmotic pressure will continue to be a limiting factor in the effectiveness and stability of cellularly-inspired energy relevant materials.

Principles of Biomolecular Network Design

Networks of biomolecular unit cells are proposed as a new type of biologically inspired intelligent materials. These materials are derived from natural cellular mechanics and aim to improve current biologically-inspired technologies by recreating the desired systems from the basic building block of the natural world; the cell. The individual biomolecular unit cell is able to replicate natural cellular abilities through a combination of lipid bilayer membranes containing embedded proteins and peptides. While individual unit cells offer an ideal testing environment for demonstrating proofs of concept, more advanced abilities require larger networks, utilizing cell-to-cell interactions.The cell-to-cell interactions often involve multiple modes of communication, which have been identified for this paper as primarily electrical, chemical, and mechanical phenomenon. Previous modeling efforts have incorporated the electrical portion through equivalent circuit models, but these lack the ability to fully explain some of the network characteristics. A new formulation is presented here to illustrate how these three classes of phenomenon may be coupled to achieve various engineering design goals.Copyright

Network modeling of membrane-based artificial cellular systems

Computational models are derived for predicting the behavior of artificial cellular networks for engineering applications. The systems simulated involve the use of a biomolecular unit cell, a multiphase material that incorporates a lipid bilayer between two hydrophilic compartments. These unit cells may be considered building blocks that enable the fabrication of complex electrochemical networks. These networks can incorporate a variety of stimuli-responsive biomolecules to enable a diverse range of multifunctional behavior. Through the collective properties of these biomolecules, the system demonstrates abilities that recreate natural cellular phenomena such as mechanotransduction, optoelectronic response, and response to chemical gradients. A crucial step to increase the utility of these biomolecular networks is to develop mathematical models of their stimuli-responsive behavior. While models have been constructed deriving from the classical Hodgkin-Huxley model focusing on describing the system as a combination of traditional electrical components (capacitors and resistors), these electrical elements do not sufficiently describe the phenomena seen in experiment as they are not linked to the molecular scale processes. From this realization an advanced model is proposed that links the traditional unit cell parameters such as conductance and capacitance to the molecular structure of the system. Rather than approaching the membrane as an isolated parallel plate capacitor, the model seeks to link the electrical properties to the underlying chemical characteristics. This model is then applied towards experimental cases in order that a more complete picture of the underlying phenomena responsible for the desired sensing mechanisms may be constructed. In this way the stimuli-responsive characteristics may be understood and optimized.

Combined Modeling of Bilayer Networks for Sensing Applications

The bilayer lipid membrane (BLM) is a naturally occurring thin layer of phospholipid molecules that surrounds cellular systems. The membrane operates as a near-impermeable barrier allowing for the generation of membrane potentials across the layer through changes in ionic concentrations. This membrane is required for regular cell function ranging from storing energy to passing signals. Engineering advancements have allowed for the rapid creation of artificial bilayer membranes, and these membranes are currently considered for many biomimetic applications.The application of interest for this paper is the further development of these cellular systems for sensing applications. This will be accomplished through a combined fluid-bilayer model, allowing for study of the bilayer transduction properties at both high and low frequencies. Several approaches are discussed and applied to multiple cell systems with or without embedded voltage-dependent ion pores. Finally the results are studied and evidence is presented for the development of a new molecular model for cellular systems combining chemical, electrical, and mechanical stimuli.Copyright