Browsing by Author "Chen, Hueih-Min"

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Chip-based protein-protein interaction studied by atomic force microscopy
(2012-10-01)
Kao, Feng-Sheng
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Ger, Waylon
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Pan, Yun-Ru
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Yu, Hui-Chen
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Hsu, Ray-Quen
In this article, a technique for accurate direct measurement of protein-to-protein interactions before and after the introduction of a drug candidate is developed using atomic force microscopy (AFM). The method is applied to known immunosuppressant drug candidate Echinacea purpurea derived cynarin. T-cell/CD28 is on-chip immobilized and B-cell/CD80 is immobilized on an AFM tip. The difference in unbinding force between these two proteins before and after the introduction of cynarin is measured. The method is described in detail including determination of the loading rates, maximum probability of bindings, and average unbinding forces. At an AFM loading rate of 1.44 x 104?pN/s, binding events were largely reduced from 61?+/- 5% to 47?+/-?6% after cynarin introduction. Similarly, maximum probability of bindings reduced from 70% to 35% with a blocking effect of about 35% for a fixed contact time of 0.5?s or greater. Furthermore, average unbinding forces were reduced from 61.4 to 38.9?pN with a blocking effect of similar to 37% as compared with similar to 9% by SPR. AFM, which can provide accurate quantitative measures, is shown to be a good method for drug screening. The method could be applied to a wider variety of drug candidates with advances in bio-chip technology and a more comprehensive AFM database of protein-to-protein interactions. Biotechnol. Bioeng. 2012; 109: 24602467. (c) 2012 Wiley Periodicals, Inc.
Compact dimension of denatured states of staphylococcal nuclease
(2008-08-15)
Chow, C. -Y.
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Wu, Ming-Chya
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Fang, Huey-Jen
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Hu, Chin-Kun
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Chen, Hueih-Min
Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5 '-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 angstrom while it is 20.4 angstrom for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model. No reversible arrow D-1 reversible arrow D-2 reversible arrow D-3 (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.
Efficacy verification and microscopic observations of an anticancer peptide, CB1a, on single lung cancer cell
(2012-12-01)
Kao, Feng-Sheng
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Pan, Yun-Ru
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Hsu, Ray-Quen
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Chen, Hueih-Min
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機械工程學系
In this work, we introduce a new customized anti-lung cancer peptide, CB1a, with IC50 of about 25.0 +/- 1.6 mu M on NCI-H460 lung cancer cells. Using a multi-cellular tumor spheroid (MCTS) model, results show that CB1a is potent in preventing the growth of lung cancer tumor-like growths in vitro. Additionally, atomic force microscopy (AFM) was used to examine cell surface damage of a single cancer. The mechanism for cell death under CB1a toxicity was verified as being largely due to cell surface damage. Moreover, with a treatment dosage of CB1a at 25 mu M, Young's module (E) shows that the elasticity and stiffness of cancer cell decreased with time such that the interaction time for a 50% reduction of E (IT50) was about 7.0 min. This new single-cell toxicity investigation using IT50 under AFM assay can be used to separately verify drug efficacy in support of the traditional IC50 measurement in bulk solution. These results could be of special interest to researchers engaged in new drug development. (C) 2012 Elsevier B.V. All rights reserved.
Efficiency optimisation of proteins on a chip
(2015-01-01)
Wu, Wei-jen
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Huang, Hsuan-yu
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Hsu, Wei-Yeh
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Hsu, Ray-Quen
;
Chen, Hueih-Min
This study elucidates that the protein reorientation on a chip can be changed by an external electric field (EEF) and optimised for achieving strong effective binding between proteins. Protein A and its binding protein immunoglobulin G (IgG) were used as an example, in addition to an anticancer peptide (CB1a) and its antibody (anti-CB1a). The binding forces (BFs) were measured by atomic force microscopy (AFM) with EEFs applied at different angles (EEF degrees). The optimal angle (OA) of the EEF (OAEEF degrees) corresponding to the maximum binding force (BFmax) was obtained. The results showed that the BFmax values between IgG/Protein A and anti-CB1a/CB1a were 6424.2 +/- 195.3 pN (OAEEF degrees = 45 degrees) and 729.1 +/- 33.2 pN (OAEEF degrees = 22.5 degrees), respectively. Without an EEF, the BF values were only 730.0 +/- 113.9 pN and 337.3 +/- 35.0 pN, respectively. Based on these observations, we concluded that the efficient optimisation of protein-protein interaction on a chip is essential. This finding is applicable to the industrial fabrication of all protein chips.
Parallel synthesis and biological evolution of quinic acid derivatives as immuno-suppressing agents against T-cell receptors
(2015-01-01)
Huang, Chih-Yu
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Chen, Li-Hsun
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Huang, Hsuan-Yu
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Kao, Feng-Sheng
;
Lee, Yun-Ta
A simple protocol for the synthesis of quinic acid derivatives was established and their biological evolution against T-cells is studied. Results showed that one of the derivatives, Cyn-1324, has low toxicity on T-cells and a high effect on reducing Signal 2 of T-cell immune responses. In vitro binding measurements of atomic force spectroscopy further indicated that the blocking effect of Cyn-1324 between CD28 and CD80 was about 31 + 4%. In vivo animal tests also confirmed that Cyn-1324 can reduce the allergic responses from ovalbumin-induced mice with little toxicity. Based on these observations, Cyn-1324 can be a mild immuno-suppressive candidate for future drug development.
Thermostability of the N-Terminal RNA-Binding Domain of the SARS-CoV Nucleocapsid Protein: Experiments and Numerical Simulations
(2009-03-04)
Fang, Huey-Jen
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Chen, Yong-Zhong
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Li, Mai Suan
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Wu, Ming-Chya
;
Chang, Chun-Ling
Differential scanning calorimetry, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and numerical simulations were used to study the thermostability of the N-terminal RNA-binding domain (RBD) of the SARSCoV nucleocapsid protein. The transition temperature of the RBD in a mixing buffer, composed of glycine, sodium acetate, and sodium phosphate with 100 mM sodium chloride, at pH 6.8, determined by differential scanning calorimetry and circular dichroism, is 48.74 degrees C. Experimental results showed that the thermal-induced unfolding-folding transition of the RBD follows a two-state model with a reversibility >90%. Using a simple Go-like model and Langevin dynamics we have shown that, in agreement with our experiments, the folding of the RBD is two-state. Theoretical estimates of thermodynamic quantities are in reasonable agreement with the experiments. Folding and thermal unfolding pathways of the RBD also were experimentally and numerically studied in detail. It was shown that the strand 01 from the N-terminal folds last and unfolds first, while the remaining beta-strands fold/unfold cooperatively.