Institute of Bioinformatics and Structural Biology

Hsinchu, Taiwan

Institute of Bioinformatics and Structural Biology

Hsinchu, Taiwan
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Liu T.-H.,Institute of Bioinformatics and Structural Biology | Huang Y.-T.,Institute of Bioinformatics and Structural Biology | Cheng H.-W.,National Chiao Tung University | Chen Y.-W.,Institute of Bioinformatics and Structural Biology | And 4 more authors.
Journal of Physical Chemistry C | Year: 2015

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. © 2015 American Chemical Society.


Chen F.-F.,Institute of Bioinformatics and Structural Biology | Lin W.-H.,Institute of Bioinformatics and Structural Biology | Lin S.-C.,Institute of Bioinformatics and Structural Biology | Kuo J.-H.,Institute of Bioinformatics and Structural Biology | And 7 more authors.
Glycobiology | Year: 2012

Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (KD) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan- proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70 of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin. © The Author 2012.


Hsu S.-H.,Chang Gung University | Lo Y.-Y.,Chang Gung University | Liu T.-H.,Institute of Bioinformatics and Structural Biology | Pan Y.-J.,Institute of Bioinformatics and Structural Biology | And 6 more authors.
Journal of Biological Chemistry | Year: 2015

Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H+-pyrophosphatase (EC 3.6.1.1) from Vigna radiata . H+-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H+-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H+-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H+-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H+-pyrophosphatase in the presence and absence of substrate and its analog. © 2015 by The American Society for Biochemistry and Molecular Biology Inc.


Kuo Z.-Y.,Laboratory of Control and Systems Biology | Chuang Y.-J.,Institute of Bioinformatics and Structural Biology | Chao C.-C.,Institute of Bioinformatics and Structural Biology | Liu F.-C.,Institute of Bioinformatics and Structural Biology | And 3 more authors.
Journal of Innate Immunity | Year: 2013

Candida albicans infections and candidiasis are difficult to treat and create very serious therapeutic challenges. In this study, based on interactive time profile microarray data of C. albicans and zebrafish during infection, the infection-related protein-protein interaction (PPI) networks of the two species and the intercellular PPI network between host and pathogen were simultaneously constructed by a dynamic interaction model, modeled as an integrated network consisting of intercellular invasion and cellular defense processes during infection. The signal transduction pathways in regulating morphogenesis and hyphal growth of C. albicans were further investigated based on significant interactions found in the intercellular PPI network. Two cellular networks were also developed corresponding to the different infection stages (adhesion and invasion), and then compared with each other to identify proteins from which we can gain more insight into the pathogenic role of hyphal development in the C. albicans infection process. Important defense-related proteins in zebrafish were predicted using the same approach. The hyphal growth PPI network, zebrafish PPI network and host-pathogen intercellular PPI network were combined to form an integrated infectious PPI network that helps us understand the systematic mechanisms underlying the pathogenicity of C. albicans and the immune response of the host, and may help improve medical therapies and facilitate the development of new antifungal drugs. Copyright © 2013 S. Karger AG, Basel.


Liao C.-C.,National Yang Ming University | Chen Y.-W.,Institute of Bioinformatics and Structural Biology | Jeng T.-L.,Agricultural Research Institute | Li C.-R.,Shih Chien University | Kuo C.-F.,Shih Chien University
Journal of Agricultural and Food Chemistry | Year: 2013

A high level of intake of vegetables is strongly associated with the prevention of chronic diseases. Because post-translational modifications (PTMs) have been shown to be the important biomarkers of the change in physiological functions, this study aimed to explore the changes in PTMs of plasma proteins when purple sweet potato (PSP), a root vegetable, was incorporated into the daily diet. Male Syrian hamsters were maintained on a rice diet (50% rice) or PSP diet (25% rice and 25% PSP) for 12 weeks. Plasma proteins were fractionated by electrophoresis, digested by trypsin, and then separated by nano-liquid chromatography and tandem mass spectrometry. The TurboSequest algorithm was used to identify peptide sequence against the hamster database in Universal Proteins Resource Knowledgebase, and in-house PTM finder programs were used for identification and quantification of PTMs. The results indicated that 95 plasma proteins were identified and 28 PTM sites on 26 of these 95 proteins were affected by consumption of PSP (p < 0.05). Methylation accounted for the largest percentage of affected modifications (35.71%). This study also showed that incorporation of purple sweet potato into the diet significantly lowered blood and liver lipids (p < 0.05). The results of this study provide a basis for prospective studies evaluating the effects of dietary intervention on modifications of proteins. © 2013 American Chemical Society.


Kuo J.-H.,Institute of Bioinformatics and Structural Biology | Chen Y.-P.,Institute of Bioinformatics and Structural Biology | Liu J.-S.,Institute of Bioinformatics and Structural Biology | Dubrac A.,University of Bordeaux 1 | And 9 more authors.
Journal of Biological Chemistry | Year: 2013

Background: CXCL4L1 is a highly potent anti-angiogenic and anti-tumor chemokine, and its structural information is unknown. Results: CXCL4L1 x-ray structure is determined, and it reveals a previously unrecognized chemokine structure adopting a novel C-terminal helix conformation. Conclusion: The alternative helix conformation enhances the anti-angiogenic activity of CXCL4L1 by reducing the glycosaminoglycan binding ability. Significance: Chemokine C-terminal helix orientation is critical in regulating their functions. Copyright © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


Huang Y.-T.,Institute of Bioinformatics and Structural Biology | Liu T.-H.,Institute of Bioinformatics and Structural Biology | Lin S.-M.,Institute of Bioinformatics and Structural Biology | Chen Y.-W.,Institute of Bioinformatics and Structural Biology | And 5 more authors.
Journal of Biological Chemistry | Year: 2013

Homodimeric proton-translocating pyrophosphatase (H+- PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H+-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H+-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H+-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H+-PPase proton translocation.Aworking model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H+-PPase upon substrate binding. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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