蛋白質摺疊之理論研究： 從實驗本質以及分子動力學模擬來探討現象模型的設計

Theoretical Studies of Protein Folding: Experimental and Molecular Dynamics Insights into the Design of Phenomenological Models

蔡旻燁(Min-Yeh Tsai)

指導教授 ： 林聖賢

國立臺灣大學/理學院/化學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.02450

摘要

一般來說在分析蛋白質摺疊的熱力學性質時，不同實驗方法會得到不一致的結果。例如使用不同的光譜方法( spectroscopy methods )以及示差掃描量熱法( differential scanning calorimetry, DSC )等來分析蛋白質摺疊的自由能。這是因為有些實驗探測的是蛋白質其局部( local )的性質，而有些實驗量測的是蛋白質整體( global )。這類與實驗探針( probe )選擇相關的議題，其理論基礎仍尚未建立。本論文的目的即是參照不同實驗方法的結果以期能理解各實驗方法的特性，並設計符合各實驗結果的現象模型。我的模型是從統計力學的觀點來解釋不同實驗方法所得到的結果。
論文的第一部分先以雙態模型( two-state model )探討細胞色素( cytochrome c, Cyt c )其摺疊的熱力學性質( 例如自由能變化等 )，主要使用的實驗技術有UV-vis吸收光譜( UV-vis absorption spectroscopy )、圓二色光譜( circular dichroism )、螢光光譜( fluorescence spectroscopy )以及示差量熱掃描儀( DSC )。結果顯示，由光譜方法分析的熱力學性質是屬於局部的，而由DSC 所量測到的是屬於蛋白質整體的性質。這與氫離子交換實驗( HX experiment )的結果一致。Englander 教授用HX實驗發現了摺疊子( foldon )的存在，並用此觀念來解釋蛋白質局部以及整體的性質。我們根據此觀念提出了一個簡化的近似拉鍊模型( zipper-like model ) 以解釋Cyt c 摺疊的熱力學以及動力學性質。此外，我使用分子動力學模擬來觀察 Cyt c 其摺疊子的巨觀行為。我的結果支持 Cyt c 的摺疊機制遵循古典的蛋白質摺疊路徑。
論文的第二部分探討一小段β-髮夾胜肽其摺疊的熱力學性質。我用微觀的方法來解釋β-髮夾胜肽與摺疊子的關係。我提出一個統計力學模型，主要是Wako-Saito-Munoz-Eaton (WSME) 模型的修改版本。此修改後的模型特別強調局部摺疊熱力學性質的重要。我們發展了計算此模型配分函數 ( partition function )的方法，稱為 contact-pair treatment。此外，分子動力學模擬的結果亦支持我們在局部熱力學性質上的假設。這表示我們所提出的模型確實能提供摺疊子行為在統計力學上的基礎。這項研究或許能在詮釋蛋白質局部的熱力學行為以及蛋白質摺疊相關問題上有所幫助。

關鍵字

蛋白質摺疊 ； 熱力學 ； 動力學 ； 拉鍊模型 ； Wako-Sait&ocirc ； -Mu&ntilde ； oz-Eaton (WSME) 模型 ； 分子動力學模擬 ； 光譜 ； 示差量熱掃描 ； 細胞色素 ； GB1 beta-髮夾胜肽摺疊子 ； 實驗探針選擇性 ； 局部熱力學行為

並列摘要

Analyzing experimental data of thermodynamic properties of proteins reveals large discrepancies between data obtained using different experimental methods, for example, those obtained by spectroscopy methods and those by calorimetry. The interpretation is that some experimental methods probe local or site-specific properties of proteins, while others probe global properties. However, the theoretical foundation of this probe-dependent issue has not yet been established. The aim of this thesis attempts to investigate the phenomenological models, which are designed according to insight from experimental results. These models aim to explain the different results from different experimental methods from a statistical mechanics point of view.
In the first part of the thesis, the thermodynamic properties (e.g., the free energy change) of cytochrome c (Cyt c) were analyzed using the thermodynamic two-state model via absorption spectroscopy (Abs), circular dichroism (CD), fluorescence spectroscopy (Flu) and differential scanning calorimetry (DSC). These results showed that the thermodynamic properties measured by spectroscopic methods are local properties, and those measured by DSC are, however, global properties. This also confirms the existence of macro-units in Cyt c, identified as foldons by Englander’s group using hydrogen exchange (HX) experiments. A simplified zipper-like model was accordingly proposed to describe the folding-unfolding thermodynamics and kinetics of Cyt c. In addition, molecular dynamics (MD) simulations were performed to detect the macro- (or foldon) behavior of Cyt c from microscopic molecular motions. The result was in line with a thermodynamically observed sequential folding mechanism, supporting that Cyt c folding occurs in accordance with the classical pathway concept.
In the second part of the thesis, the thermodynamic behavior and its related properties (e.g., the folding fraction and free energy change) of a β-hairpin peptide (GB1 C-terminal β-hairpin) were investigated in order to understand the folding of the basic structural motif. I also defined the foldon behavior from a microscopic point of view. Based on this view, I proposed a modified Wako-Saito-Munoz-Eaton (WSME) model, designed with site-dependent properties of proteins in mind. A mathematical technique (contact-pair treatment) was accordingly developed for facilitating the calculation of the partition function. In addition, I performed MD simulations and the results showed the same site-dependent characteristics. Our results showed that the proposed model provides a statistical mechanical foundation for the foldon behavior, and may be generally useful in the interpretation of site-dependent properties of proteins as well as the study of protein folding.

並列關鍵字

protein folding ； thermodynamics ； kinetics ； zipper-like model ； Wako-Sait&ocirc ； -Mu&ntilde ； oz-Eaton (WSME) model ； molecular dynamics (MD) simulation ； spectroscopy ； differential scanning calorimetry (DSC) ； cytochrome c ； GB1 C-terminal beta-hairpin ； foldon ； probe-dependent ； site-dependent

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