Browsing by Author "Pan, Yun-Ru"

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Chip-based protein-protein interaction studied by atomic force microscopy
(2012-10-01)
Kao, Feng-Sheng
;
Ger, Waylon
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Pan, Yun-Ru
;
Yu, Hui-Chen
;
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.
Efficacy verification and microscopic observations of an anticancer peptide, CB1a, on single lung cancer cell
(2012-12-01)
Kao, Feng-Sheng
;
Pan, Yun-Ru
;
Hsu, Ray-Quen
;
Chen, Hueih-Min
;
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.
Solution structure and phospho-PmrA recognition mode of PmrD from Klebsiella pneumoniae
(2010-12-01)
Luo, Shih-Chi
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Lou, Yuan-Chao
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Cheng, Hsin-Yao
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Pan, Yun-Ru
;
Peng, Hwei-Ling
In bacteria, the two-component system (TCS) is the most prevalent for sensing and transducing the environmental signals into the cell. In Salmonella, the small basic protein PmrD is found to protect phospho-PmrA and prolong the expression of PmrA-activated genes. In contrast, Escherichia coli PmrD fails to protect phospho-PmrA. Here, we show that Klebsiella pneumoniae PmrD (KP-PmrD) can inhibit the dephosphrylation of phospho-PmrA, and the interaction between KP-PmrD and the N-terminal receiver domain of PmrA (PmrA(N)) is much stronger in the presence than in the absence of the phosphoryl analog beryllofluoride (BeF(3)(-)) (K(D) = 1.74 +/- 0.81 mu M vs. K(D) = 236 +/- 48 mu M). To better understand the molecular interactions involved, the solution structure of KP-PmrD was found to comprise six beta-strands and a flexible C-terminal alpha-helix. Amide chemical shift perturbations of KP-PmrD in complex with BeF(3)(-)-activated PmrA(N) suggested that KP-PmrD may undergo a certain conformational rearrangement on binding to activated PmrA(N). Saturation transfer experiments revealed the binding surface to be located on one face of the beta-barrel. This finding was further verified by in vivo polymyxin B susceptibility assay of the mutants of KP-PmrD. The phospho-PmrA recognition surface of KP-PmrD, which involves two KP-PmrD proteins in complex with an activated-PmrA(N) dimer, is suggested to be a contiguous patch consisting of Trp3, Trp4, Ser23, Leu26, Glu27, Met28, Thr46, Leu48, Ala49, Asp50, Ala51, Arg52,Ile65, Asn67, Ala68, Thr69, His70, Tyr71, Ser73 and Glu74. Our study furthers the understanding of how PmrD protects phopho-PmrA in the PmrAB TCS. (C) 2010 Elsevier Inc. All rights reserved.