Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity

Watanabe, Rikiya; Soga, Naoki; Fujita, Daishi; Tabata, Kazuhito V.; Yamauchi, Lisa; Kim, Soo Hyeon; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Suga, Hiroaki; Noji, Hiroyuki

doi:10.1038/ncomms5519

Cited by 106 publications

(143 citation statements)

References 42 publications

(49 reference statements)

Supporting

Mentioning

137

Contrasting

Order By: Relevance

“…Thus, unlike other structures made by using painting, flowing water/oil/water, and microjetting methods, [14,42,43] they do not denaturize the proteins reconstituted in the membrane of the structures. Thus, unlike other structures made by using painting, flowing water/oil/water, and microjetting methods, [14,42,43] they do not denaturize the proteins reconstituted in the membrane of the structures.…”

Section: Biofunctionality Assay Of the Generated Structuresmentioning

confidence: 98%

Generation of Solvent‐Free 3D Lipid Structure Arrays on High Aspect Ratio Si Microwell Substrate

Han

Kwak

Kang

et al. 2019

Adv Materials Inter

View full text Add to dashboard Cite

the substrate (≈100 nm to ≈100 µm in diameter), which are more similar to cells with an internal space surrounded by a cell membrane. In this case, although the lipid membrane is slightly more fragile than the lipid bilayer that is deposited directly or tethered on the substrate, it is suitable for assessing the activity of the ion channel through an ion channel embedded in the lipid layer. [12,13] Several approaches have been used to form such a suspended lipid bilayer (black lipid membranes), including painting, [14] folding, [15] liposome bursting, [16] solution flowing, [17] and tip-dip methods. [18] However, such processes do not meet the requirements for the mass production of robust, synthetic, lipid-based devices. In particular, commonly used painting or solution flow methods are not solvent free in the lipid layers, which is very harmful to the stability of membrane proteins embedded in the lipid layers. [19][20][21] Another important consideration is that almost all solid supported membrane devices consist of a 2D, planar-type lipid membrane structure. Therefore, the reduced size of the aperture in such planar-type devices that are used to increase membrane stability causes a severe reduction of the bilayer lipid membrane area, which appears to be the main disadvantage for the reconstitution of many proteins. [22,23] In the studies of cellular membranes through fluorescent observation using confocal microscopy or fluorescent microscopes, it is expected that a 3D lipid structure array on a chip is very powerful because the structure array has the same focal plane. Additional advantages of 3D lipid structure include the capability of multiplexing access and the improved binding characteristics on the membranes that have the proper curvature, i.e., curvature that is similar to that of the surface of cells. However, although giant unilamellar vesicle (GUV) arrays attached to substrate by a linker have been reported as a platform for various applications such as a bioreactor and screenings of single molecule reaction and lipid-protein interaction, [24] to date, reports of 3D lipid structure arrays being formed directly on solid supports have been rare. Kang et al. presented the electroformation of a controlled-size, 3D GUV array using a hydrogel stamping method on an indium tin oxide (ITO) glass substrate for floating GUV formation by detaching them from the substrate. [25] In addition to this generation of uniform-size, floating GUVs, Kang et al. mentioned the possibility of applying a form of GUV array attached to the substrate Artificial lipid membranes are versatile platforms that are used extensively in biological assays and sensing applications. Particularly, a 2D bilayer lipid membrane (BLM) has been focused on over the last several decades as it can be formed easily on solid supports by various methods. However, 3D lipid structures with structural advantages, such as large surface area that can accommodate a number of proteins and steric conformation that can react with target molecules efficiently, ...

show abstract

Section: Biofunctionality Assay Of the Generated Structuresmentioning

confidence: 98%

Generation of Solvent‐Free 3D Lipid Structure Arrays on High Aspect Ratio Si Microwell Substrate

Han

Kwak

Kang

et al. 2019

Adv Materials Inter

View full text Add to dashboard Cite

show abstract

“…In vivo, F 1 forms F o F 1 -ATP synthase and synthesizes ATP by coupling with a rotary motion driven by F o . Recently, the rotary motion and proton translocation of the F o F 1 complex on a lipid bilayer membrane was visualized at the singlemolecule level (7,8,59). For further understanding of the effect of the ε subunit on catalysis at physiological conditions, verification by single-molecule measurement of the F o F 1 complex is desirable in the future.…”

Section: Fmentioning

confidence: 99%

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase

Watanabe

Genda

Kato-Yamada

et al. 2018

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

F-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.

show abstract

“…Therefore, the biological nanopore becomes the only pathway between the two aqueous phases separated by the bilayer. Diverse types of lipid bilayer platforms have been developed till date [5][6][7][8][9][10][11][12][13][14][15][16]. An example is the double-well chip, in which a bilayer is formed between a pair of aqueous droplets in lipid-dispersed oil [5,6].…”

Section: Lipid Bilayer-based Sensorsmentioning

confidence: 99%

Membrane protein-based biosensors

Misawa

Osaki

Takeuchi

2018

J. R. Soc. Interface.

View full text Add to dashboard Cite

This review highlights recent development of biosensors that use the functions of membrane proteins. Membrane proteins are essential components of biological membranes and have a central role in detection of various environmental stimuli such as olfaction and gustation. A number of studies have attempted for development of biosensors using the sensing property of these membrane proteins. Their specificity to target molecules is particularly attractive as it is significantly superior to that of traditional human-made sensors. In this review, we classified the membrane protein-based biosensors into two platforms: the lipid bilayer-based platform and the cell-based platform. On lipid bilayer platforms, the membrane proteins are embedded in a lipid bilayer that bridges between the protein and a sensor device. On cell-based platforms, the membrane proteins are expressed in a cultured cell, which is then integrated in a sensor device. For both platforms we introduce the fundamental information and the recent progress in the development of the biosensors, and remark on the outlook for practical biosensing applications.

show abstract

Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity

Cited by 106 publications

References 42 publications

Generation of Solvent‐Free 3D Lipid Structure Arrays on High Aspect Ratio Si Microwell Substrate

Generation of Solvent‐Free 3D Lipid Structure Arrays on High Aspect Ratio Si Microwell Substrate

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase

Membrane protein-based biosensors

Contact Info

Product

Resources

About