Browsing by Author "Chen, Yen-Wei"

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Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase
(2013-12-01)
Lee, Ching-Hung
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Chen, Yen-Wei
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Huang, Yun-Tzu
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Pan, Yih-Jiuan
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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.
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
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Chen, Yen-Wei
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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.