Browsing by Author "Wu, Ming-Chya"

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Compact dimension of denatured states of staphylococcal nuclease
(2008-08-15)
Chow, C. -Y.
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Wu, Ming-Chya
;
Fang, Huey-Jen
;
Hu, Chin-Kun
;
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.
Hydrophobic condensation and modular assembly model of protein folding
(2008-07-01)
Tsong, Tian-Yow
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Hu, Chin-Kun
;
Wu, Ming-Chya
;
交大名義發表
;
National Chiao Tung University
Despite several decades of intense study, protein folding problem remains elusive. In this paper, we review current knowledge and the prevailing thinking in the field, and summarize our work on the in vitro folding of a typical small globular protein, staphylococcal nuclease (SNase). Various thermodynamic and kinetic methods have been employed to determine the energetic and construct the energy landscape of folding. Data presented include, but not limit to, the identification of intermediate states, time courses of their spread and convergence on the landscape, and finally the often ignored step, the refinement of the overall conformation and hence the activation of the enzyme. Our goal is to have a complete perspective of the folding process starting from its initial unfolded state to the fully active native state. Analysis leads to these findings: the folding starts with the condensation of the hydrophobic side chains in different locales of the peptide chain. The newly forged hydrophobic environment facilitates formation of helix- and sheet-like frameworks at different domains. Consolidation and inter-docking of these frameworks or domains then stabilizes the overall conformation and refines the structure to activate the enzyme. Based on these observations we favor folding-by-parts and propose a modular assembly model for the in vitro folding of SNase. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
Surface electromyography analysis of blepharoptosis correction by transconjunctival incisions
(2016-06)
Tu, Lung-Chen
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Wu, Ming-Chya
;
Chu, Hsueh-Liang
;
Chiang, Yi-Pin
;
Kuo, Chih-Lin
Upper eyelid movement depends on the antagonistic actions of orbicularis oculi muscle and levator aponeurosis. Blepharoptosis is an abnormal drooping of upper eyelid margin with the eye in primary position of gaze. Transconjunctival incisions for upper eyelid ptosis correction have been a welldeveloped technique. Conventional prognosis however depends on clinical observations and lacks of quantitatively analysis for the eyelid muscle controlling. This study examines the possibility of using the assessments of temporal correlation in surface electromyography (SEMG) as a quantitative description for the change of muscle controlling after operation. Eyelid SEMG was measured from patients with blepharoptosis preoperatively and postoperatively, as well as, for comparative study, from young and aged normal subjects. The data were analyzed using the detrended fluctuation analysis method. The results show that the temporal correlation of the SEMG signals can be characterized by two indices associated with the correlation properties in short and long time scales demarcated at 3 ms, corresponding to the time scale of neural response. Aging causes degradation of the correlation properties at both time scales, and patient group likely possess more serious correlation degradation in long-time regime which was improved moderately by the ptosis corrections. We propose that the temporal correlation in SEMG signals may be regarded as an indicator for evaluating the performance of eyelid muscle controlling in postoperative recovery. (C) 2016 Elsevier Ltd. All rights reserved.
Thermostability of the N-Terminal RNA-Binding Domain of the SARS-CoV Nucleocapsid Protein: Experiments and Numerical Simulations
(2009-03-04)
Fang, Huey-Jen
;
Chen, Yong-Zhong
;
Li, Mai Suan
;
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.