Browsing by Author "Pan, Rong-Long"

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Formation of suspended bilayer lipid membrane between electrowetting-driven encapsulated droplets
(2014-09-01)
Fan, Shih-Kang
;
Chen, Ching-Wen
;
Lin, Yi-Ying
;
Chen, Li-Chi
;
Tseng, Fan-Gang
Using limited lipid molecules held by two water-core/oil-shell encapsulated droplets, we formed an optically observable bilayer lipid membrane (BLM) across a microfabricated aperture whose dimension was precisely determined and easily scalable. For the minute volume of a 0.1 mu L oil (n-decane) shell encapsulating a 1.5 mu L water core droplet, only 0.2 to 2.8 nmol or 0.17 to 2.4 mu g lipid was required. Microscopes and electrophysiological measurements were performed on the parallel-plate device with three major steps demonstrated: (1) manipulating self-assembled lipid monolayers at the water-oil interface of the encapsulated droplets by electrowetting-on-dielectric (EWOD), (2) forming a BLM by bringing two lipid monolayers towards the aperture and thinning down the oil film between them by Young-Laplace pressure, and (3) incorporating membrane-bound nanopores, alpha-hemolysin (alpha HL), on the BLM. This study shows the influence of the lipid concentration to the interfacial tension and EWOD. Plateau-Gibbs border and black membrane area of the BLM were optically observed, while trans-membrane electrophysiological signals were electrically recorded from a pair of Ag/AgCl electrodes. (C) 2014 AIP Publishing LLC.
Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase
(2013-12-01)
Lee, Ching-Hung
;
Chen, Yen-Wei
;
Huang, Yun-Tzu
;
Pan, Yih-Jiuan
;
Lee, Chien-Hsien
H+-translocating pyrophosphatase (H+-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH(+)-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H+-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H+-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH(+)-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K+-mediated stimulation of H+-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase.
Incorporating Support Vector Machine for Identifying Protein Tyrosine Sulfation Sites
(2009-11-30)
Chang, Wen-Chi
;
Lee, Tzong-Yi
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Shien, Dray-Ming
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Hsu, Justin Bo-Kai
;
Horng, Jorng-Tzong
Tyrosine sulfation is a post-translational modification of many secreted and membrane-bound proteins. It governs protein-protein interactions that are involved in leukocyte adhesion, hemostasis, and chemokine signaling. However, the intrinsic feature of sulfated protein remains elusive and remains to be delineated. This investigation presents SulfoSite, which is a computational method based on a support vector machine (SVM) for predicting protein sulfotyrosine sites. The approach was developed to consider structural information such as concerning the secondary structure and solvent accessibility of amino acids that surround the sulfotyrosine sites. One hundred sixty-two experimentally verified tyrosine sulfation sites were identified using UniProtKB/SwissProt release 53.0. The results of a five-fold cross-validation evaluation suggest that the accessibility of the solvent around the sulfotyrosine sites contributes substantially to predictive accuracy. The SVM classifier can achieve an accuracy of 94.2% in fivefold cross validation when sequence positional weighted matrix (PWM) is coupled with values of the accessible surface area (ASA). The proposed method significantly outperforms previous methods for accurately predicting the location of tyrosine sulfation sites. (C) 2009 Wiley Periodicals, Inc. J Comput Chem 30: 2526-2537, 2009
Pore-Spanning Lipid Membrane under Indentation by a Probe Tip: A Molecular Dynamics Simulation Study
(2011-10-04)
Huang, Chen-Hsi
;
Hsiao, Pai-Yi
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Tseng, Fan-Gang
;
Fan, Shih-Kang
;
Fu, Chien-Chung
We study the indentation of a free-standing lipid membrane suspended over a nanopore on a hydrophobic substrate by means of molecular dynamics simulations. We find that in the course of indentation the membrane bends at the point of contact and the fringes of the membrane glide downward intermittently along the pore edges and stop gliding when the fringes reach the edge bottoms. The bending continues afterward, and the large strain eventually induces a phase transition in the membrane, transformed from a bilayered structure to an interdigitated structure. The membrane is finally ruptured when the indentation goes deep enough. Several local physical quantities in the pore regions are calculated, which include the tilt angle of lipid molecules, the nematic order, the included angle, and the distance between neighboring lipids. The variations of the;e quantities reveal many detailed, not-yet-specified local structural transitions of lipid molecules under indentation. The force indentation curve is also studied and discussed. The results make a connection between the microscopic structure and the macroscopic properties and provide deep insight into the understanding of the stability of a lipid membrane spanning over nanopore.
Simple and Fast Method To Fabricate Single-Nanoparticle-Terminated Atomic Force Microscope Tips
(2013-06-27)
Cheng, Hui-Wen
;
Chang, Yuan-Chih
;
Yuan, Chi-Tsu
;
Tang, Song-Nien
;
Chang, Chia-Seng
This paper introduces a simple, yet controllable scheme to pick up a single 13 nm Au nanoparticle (Au-NP) using the tip of an atomic force microscope (AFM) probe through the application of electrical biases between the tip and the Au-NP. Transmission electron microscope (TEM) images were acquired to verify that a single Au-NP was attached to the AFM probe. We postulate that the mechanism underlying the ability to manipulate individual Au-NPs at the apex of the AFM probe tip is Coulomb interaction induced by tip bias. The AFM tip with the attached Au-NP was then used to study the interaction between a single quantum dot (QD) and the Au-NP. The blinking behavior of single colloidal CdSe/ZnS core/shell QD was significantly suppressed with the approach of the 13 nm Au-NP attached to the AFM tip.
Single Molecule Take-and-Place Technique for Positioning a Membrane Protein on a Lipid Bilayer
(2015-09-10)
Liu, Tseng-Huang
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Huang, Yun-Tzu
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Cheng, Hui-Wen
;
Chen, Yen-Wei
;
Lee, Ching-Hung
Manipulating a single protein molecule on liposomes or planar lipid bilayers is a useful technique for studying membrane-bound proteins, receptors, or ion transporters and for delicate applications including biosensor chips, drug screening, and clinical diagnoses. However, several key breakthroughs are required for success through difficult techniques such as single protein take-and-place with reasonable spatiotemporal control. In this report, a novel method was established for placing a single transmembrane protein onto a patterned lipid bilayer. A surface-functionalized 1.8 nm gold nanoparticle was first engineered at the tip of an atomic force microscope. A single transmembrane protein, H+-translocating pyrophosphatase with six-histidine residues tag, was then picked up using such a microscopy tip where its nanoparticle was modified by nickel-nitrilotriacetic acid. Two-step fluorescence bleaching observation and quantum dot blinking analysis subsequently verified successful manipulation of a single functional protein on a lipid membrane in a predetermined manner. Furthermore, the enzymatic activity from the single H+-translocating pyrophosphatase was determined, demonstrating that membrane proteins retain their functions on the lipid bilayer through a single-molecule take-and-place technique. This innovative technique overcomes current limitations and provides a single biomolecule nanomanipulation system for versatile studies of membrane-bound proteins.
The proximity between C-termini of dimeric vacuolar H(+)-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique
(2009-08-01)
Liu, Tseng-Huang
;
Hsu, Shen-Hsing
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Huang, Yun-Tzu
;
Lin, Shih-Ming
;
Huang, Tsu-Wei
Vacuolar H(+)-translocating inorganic pyrophosphatase [vacuolar H(+)-pyrophosphatase (V-PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen, at the expense of PP(i) hydrolysis, for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function, although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton-translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 +/- 0.3 nm and 26.3 +/- 4.7 nm, whereas those of the luminal protrusions were 1.2 +/- 0.1 nm and 21.7 +/- 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6xHis tags on C-terminal ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 2.2 +/- 1.4 nm. Thus, our study is the first to provide structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.
The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique
(2009-08-01)
Liu, Tseng-Huang
;
Hsu, Shen-Hsing
;
Huang, Yun-Tzu
;
Lin, Shih-Ming
;
Huang, Tsu-Wei
Vacuolar H+-translocating inorganic pyrophosphatase [vacuolar H+-pyrophosphatase (V-PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen, at the expense of PPi hydrolysis, for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function, although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton-translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 +/- 0.3 nm and 26.3 +/- 4.7 nm, whereas those of the luminal protrusions were 1.2 +/- 0.1 nm and 21.7 +/- 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6xHis tags on C-terminal ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 2.2 +/- 1.4 nm. Thus, our study is the first to provide structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.