Reversible folding of lysozyme by a quasi-static process: A first-order-like state transition

• Main Authors: Yeh-Xu cheng, 葉旭成
• Other Authors: Chia-Ching Chang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/53677346481453857105

碩士 === 國立東華大學 === 應用物理研究所 === 92 === A first-order-like state transition model is considered to be a global reaction mechanism to directly fold protein from unfolded state to its native form. In order to more well studied verify the general applicability of this mechanism, a model protein, lysozyme, was fully unfolded by 4.5 M urea, 0.1 M dithiothreitol (DTT) in pH 3 and then refolded to its native form by way of an overcritical reaction path (a quasi-static process) or by crossing over a transition boundary directly (a direct dilution process). Within this study, lysozyme was trapped in a glassy state through a direct folding path, and can be folded into its native state by further increase of the concentration of lysozyme by micro-cone. This indicated that a state transition line or boundary may exist during the direct folding path. However, lysozyme can be continuously folded from unfolded to native state by an overcritical reaction path. Four stable folding intermediates and the native lysozyme were obtained and then secondary structures, particle size distribution, thermal stability and oxidation state of disulfide bonds were analyzed by circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Raman spectroscopy, respectively. According to the results, intermediates of both reaction paths follow the theme of collapse, sequential and first-order-like state transition model. This indicated that protein folding under different reaction paths might follow the same folding mechanism. Namely, the mechanism that is revealed by overcritical folding intermediates may represent the true mechanism of protein folding. A protein folding reaction mechanism was postulated and discussed. In spite of the global interaction mechanism, the fact that an α-helix is formed prior to the β-sheet may indicate that the protein folding is initiated by local interactions.