Session D23: Membranes and Channels

Formation and Properties of Self-Assembled Nanoparticle-Supported Lipid Bilayer Probed Through Molecular Dynamics Simulations

We have carried out coarse-grained molecular dynamics (MD) simulations to study the self-assembly procedure of a system of randomly placed lipid molecules, water beads, and a nanoparticle (NP). The self-assembly results in the formation of the NPSLBL, with the self-assembly mechanism being driven by events such as the formation of small lipid clusters, merging of the lipid clusters in the vicinity of the NP to form NP-embedded vesicle with a pore, and collapsing of that pore to eventually form the equilibrated NPSLBL system . Subsequently, we quantify the properties and the configurations of this NPSLBL system. We reveal that the equilibrated self-assembled NPSLBL system demonstrates a larger number of lipid molecules occupying the outer leaflet as compared to the inner leaflet. Secondly, the thickness of the water layer entrapped between the NP and the inner leaflet show similar values as that predicted by experiments. Finally, we reveal that the diffusivity of the lipid molecules in the outer leaflet is larger than that in the inner leaflet.   

Leveraging the physics of a barbecue lighter to genetically transform living organisms

Electroporation is a powerful method for delivering small molecules (RNA, DNA, drugs) across cell membranes by application of an electric field with a specific voltage and time constant, with applications ranging from synthetic biology to drug delivery. Leveraging the piezoelectric mechanism found within a common barbeque lighter, we develop a low-cost electroporator that we call an ElectroPen. This ElectroPen device costs 23-cents, weighs 13g, utilizes a 3D-printed case, and can be applied to genetically transform E. coli bacteria. In this talk, we will discuss both the physics of a lighter, as well as the synthetic biology protocol we have developed for electroporation using this frugal device. Using high-speed videos, we will discuss how the inner components of a lighter achieve extraordinary accelerations of 3000g force to ultimately deliver voltage pulses of up to 2,000 volts with a decay constant of 5ms. Finally, we will discuss applications of this low-cost device in broadening participation in synthetic biology in high school science laboratories.   

Listening to lipid membranes

Fluctuations of giant unilamellar vesicle and cell membranes are detected with exceptional resolution. The motion of localized membrane (<1 μm cross-section) is resolved with 5 decades in both frequency (1 Hz - 100 kHz) and amplitude (0.01 nm - 100 nm). This allows to listen to surprises in underlying membrane mechanics   

Comparing microrheological methods for measuring lipid membrane viscosity

The fluidity of lipid membranes governs the motions of bound proteins and macromolecules. Despite this, measurements of the viscosity of a lipid bilayer remain challenging to perform and interpret. Two different microrheological methods for measuring bilayer viscosity have been developed in recent years, one involving tracking phase-separated domains in giant unilamellar vesicles, and the other involving tracking elliptical lipid-anchored tracer particles. The latter approach has so far been applied only to pore-spanning black lipid membranes. The membrane viscosity values obtained by these two methods differ by an order of magnitude, however, and it is unclear whether this indicates inaccuracy of one or both techniques, or whether it is due to the difference in membrane systems examined. To resolve this discrepancy, we applied both methods simultaneously to the same lipid vesicles, featuring both phase separated domains and bound elliptical beads. We show that when applied to identical systems these methods are in agreement. The elliptical tracer method is generally applicable to vesicles of arbitrary composition, and we use it to quantify the viscosity of bilayers composed of phosphatidylcholine lipids of different chain lengths.   

Regulated ensembles and lipid membranes

Cellular membranes are composed of lipid bilayers, amphiphilic molecules with polar headgroups and hydrophobic tails. Their composition is highly complex, involving hundreds of different lipid types and the regulation mechanism is still the subject of intense research. A recent experiment [1] has shown that cholesterol concentration increases with temperature in zebrafishes, as well as the demixing temperature, two results which appear to be contradictory results since cholesterol promotes mixing. Here, we show that many aspects of the zebrafish experiments can be replicated if one assumes a chemical reaction network for regulation of acyl tails. Effectively, this would mean that acyl tail saturation is loosely regulated by cells and mainly directed by cholesterol fraction. This view also explains trends seen along the secretory pathway between cholesterol concentration and acyl tail saturation.

[1] M. Burns, K. Wisser, J. Wu, I. Levental, S. L. Veatch, ”Miscibility transition temperature scales with growth temperature in a zebrafish cell line” Biophysical Journal 113 (2017)   

Stable fabrication of a various-sized nanopore by controlled dielectric breakdown in a high-pH solution for the detection of various-sized molecules

For nanopore sensing of various-sized molecules with high sensitivity, the size of the nanopore should be adjusted according to the size of each target molecule. For solid-state nanopores, a simple and inexpensive nanopore fabrication method utilizing dielectric breakdown of a membrane is widely used. This method is suitable for fabricating a small nanopore. However, it suffers two serious problems when attempting to fabricate a large nanopore: the generation of multiple nanopores and the non-opening failure of a nanopore. In this study, we found that nanopore fabrication by dielectric breakdown of a SiN membrane under high-pH conditions (pH ≥ 11.3) could overcome these two problems and enabled the formation of a single large nanopore up to 40 nm in diameter within one minute.   

Monte Carlo simulation of pH activated conformational changes of coarse-grained sodium-proton antiporters

Sodium-proton antiporters are membrane proteins present throughout the eukaryotic and prokaryotic domains that have a critical role in balancing cell pH and cell volume. The Escherichia coli antiporter NhaA has pH-dependent behavior. There have been many studies regarding the function of this antiporter (Alhadeff and Warshel, PNAS (2015) 112 (40) 12378-12383), but the conformational energy landscape of this system at varying pH is not well-known. In this study, we map the energy landscape between two pH levels for two different conformations of the wild antiporter and a mutated variant using MD with coarse-graining. By Monte Carlo simulation on the energy landscape, we show that the mutated variant changes the stability of the antiporter without blocking the transport, in agreement with experimental results (Calinescu et. al. J. Biol. Chem. (2017) 292(19) 7932–7941).   

Multichannel Flow Cell for a Nanopore Array Sensor

Solid-state nanopore has been attracting remarkable research interest owing to its extensive potential in biological sensing applications, the robustness, and the possibility of a large-scale integration. A reliability and measurement time are two important criteria for practical uses of a nanopore sensor. An integration of nanopores into an array and simultaneous measurement are an effective approach to increase accuracy and to reduce measurement time. Recently, we have developed 4×4 SiN membrane arrays. However, the flow cell in this system had a bottle neck of exploiting all 16 membranes due to a real estate issue of flow paths. Another bottle neck was the generation of the air bubble that could cause the electrical conductance error.
In this study, the mechanism of those issues is investigated. A novel 16-channel flow cell made by a 3D printer is introduced. In this flow cell, the flow paths are arranged in both parallel and orthogonal direction to the membrane surface; and the distance between the membrane surface and the flow path is adjusted to prevent the generation of the air bubble. Nanopores were successfully yielded in all 16 membranes by dielectric breakdown technique.   

Interaction of Graphene Oxide with Model Bio-membrane: Insights into the Structure of the Membrane

Graphene oxide (GO) holds a similar structure of graphene with a range of oxygen functionalities such as carboxyl groups on the edges and, the hydroxyl and epoxies on the basal plane. Presence of oxygen-containing functional groups increases its water dispersity and facilitate its applications in many bio-fields including cell imaging, drug-delivery and bio-sensing. As the cellular membrane is the first target of any foreign molecule. Hence, the mechanism of interaction of GO with this membrane is very crucial to understand for extending the future applications of graphene-based materials. In present work, the x-ray reflectivity (XRR) and grazing incidence small-angle x-ray scattering (GISAXS) techniques have been used to extract the structural details of the GO-membrane complex. XRR study from lipid multilayers has shown a distinct effect of added GO on the position and shape of Bragg peaks obtained from the smectic liquid crystalline phases. GISAXS has provided the in-plane organization of the lipid molecules in the presence of GO in the membrane. The electron density profile obtained from the XRR analysis and the lattice deformation detected by the GISAXS study has provided the detailed molecular organization of the complex.   

Detection of streptavidin-labeled DNA using solid-state nanopores for target sequence detection

In recent years, the demand for rapid and on-site target gene detection has been increasing. For example, it is important for covering the increasing GMO (Genetically-modified-organisms) testings, and for preventing the spread of infectious diseases at an early stage. The conventional techniques for target DNA sequence detection are based on PCR and qPCR. Although these techniques are widespread, they are time-consuming and will not be able to cover all required testings. By comparison, solid-state nanopores have a potential to provide a rapid and portable testing device that is electronic and does not need optics. For realizing target sequence detection with solid-state nanopores, it is a promising approach to label the target sequence with a large molecule and enlarge a current-blockade value when DNA including the target sequence passed through a nanopore. In this study, the possibility of streptavidin (SA) as a labelled molecule was investigated. As a result, we found that the passages of SA-labelled and non-labelled DNA through a nanopore can be clearly distinguished.   

Moving while you're stuck; a mechanical model of binding facilitated transport in biological systems

Binding is broadly understood in many biological processes as a mechanism to localize molecules. Binding is also used to dictate particle motion through some biopolymer filters including the nuclear pore complex, the extracellular matrix and mucus membranes. In these cases, flexible polymers transiently bind to transported molecules. Here we describe a mechanical model to probe how binding and thermal motion can enable transport. A particle experiences random forces during binding and unbinding events while being constrained by attached tethers. This model provides insight into the mechanisms and design rules involved in binding-mediated transport in both biological and synthetic polymeric systems.   

Towards high-sensitivity phase cancellation microscopy

Label-free optical imaging techniques, such as light scattering and birefringence have long been used for detection of neural activity [1]. Wide-field interferometric microscopy has also been used for this purpose, most recently by Ling et al. [2]. Current interferometric systems are limited because of low phase measurement sensitivity of 10^-3, while a sensitivity of 10^-5 is necessary.
High sensitivity phase measurements will allow single-shot optical sensing of neural action potentials via imaging of optical path length changes. Here, we present a high phase sensitivity, phase cancellation interferometry system. We want to use a deformable mirror to generate a flat phase. A Wollaston prism is employed to split the beam into two orthogonally polarized beams with phase control. These beams are recombined for interferometric detection. A state-of-the-art 1.6M electron well-depth camera will be used for recordings. We report the analysis of our system’s sensitivity and compare it to theoretical predictions.

[1] Cohen et al. Nature, 1968. [2] Ling et al., Light: Science & Applications, 2018.   

Predicting optimal parameters for ion transport through nanopores and biological channels

Understanding, predicting and optimising ionic transport properties of pores on an atomic scale remains a critical challenge to, nanotechnology and biophysics [1]. In general, pores are designed to fulfil two criteria: conduction of ions at a high rate; and the selectivity amongst ionic species. We have derived a statistical and linear response theory that calculates the occupancy and conductivity of nanopores for given parameters including: pore geometry and charge; type of competing ionic species; and bulk concentration [2]. We find resonant conduction under known conditions, resulting in the coexistence of resonance for one species and suppression for the other and can predict optimal parameters required to produce the desired function. Examples of applications to biological channels and nanopores will be discussed.

[1] Hille, B., 2001, 3rd ed. Sinauer Ass., Sunderland, M.A. & Wang. L., et. al., Nat. nanotechnology, 2017. [2] Gibby, W.A.T., et. al., "Statistical theory of mixed-valence selectivity in biological ion channels", ICNF preprint, 2019. & Gibby, W.A.T., et. al., "Theory and experiments on multi-ion permeation and selectivity in the NaChBac ion channel", FNL 18, 1940007 2019.