利用拴束圓錐模型理論分析侷限於奈米孔洞中胜肽鏈之電子自旋標記光譜

Theoretical Analysis of the Spin-label ESR Spectra for the Confined Polypeptides within Nanochannels Using Tether-in-a-cone Model

錢家隆(Chia-Lung Chien)

指導教授 ： 江昀緯

國立清華大學/理學院/化學系/碩士(2012年)

摘要

近年來，研究奈米孔洞材料應用於生物分子的生物物理現象，有許多顯著的進展。在最近SDSL-ESR的研究中發現，n3 peptide (prion protein的部份氨基酸序列142~166)，在孔洞材料MSU的侷限環境中，比起在一般含抗凍劑的 bulk solution 環境中，可以有效地降低分子翻轉運動以及分子內的側鏈運動，使得cw-ESR光譜在200 K至300 K呈更慢運動光譜，使距離分布有更佳的解析度且使n3 peptide骨架的動態(dynamic)更加明顯。本文詳細推導Tether-in-a-cone(TIAC)模型理論，並建立TIAC模型的Matlab模擬程式。更進一步地，以模擬程式進行模型研究，嘗試找出TIAC模型當中的圓錐幅度μmax、位向參數ψ1、ψ2、ζ2 等參數對於其模擬cw-ESR光譜線形變化以及距離分布變化，並找出其適宜模擬條件，主要發現圓錐幅度μmax 的縮小，會使距離分布變窄，以及位向參數ψ1、ψ2、ζ2對光譜及距離分布影響減少；同時實際模擬在孔洞材料MSU以及含抗凍劑的溶液中的n3 peptide 在144及150位點雙自旋標記cw-ESR的實驗光譜數據，發現MSU中的n3 peptide，其side chain R1圓錐幅度μmax較bulk solution中縮小，距離分布變窄，顯示出MSU的奈米侷限效應有效地降低側鏈運動，同時其α-helix及β-hairpin的位點144及150之side chain R1位向差異明顯出現，呈反平行及平行。由TIAC模型的模擬結果，認為TIAC模型位向參數變化顯示出了骨架結構以及動態的大小變化；對往後生物分子在局限孔洞材料中的ESR光譜模擬擬合，提供了可靠的物理模型，描述在孔洞材料中受局限效應影響的生物分子其結構動態；同時也提出了模型的可能改進的物理理論以及程式方向，TIAC模型可以廣泛應用至各ESR技術中。

關鍵字

電子標記光譜 ； 拴束圓錐模型 ； 奈米孔洞材料

並列摘要

Spin-labeled electron spin resonance (ESR) in recent years has been combined together with mesoporous materials and demonstrated as a new biophysical methodology. Upon encapsulation of the spin-labeled biomolecules into the nanochannel of the mesoporous materials, the nano-confinement effects were proved to be effective in reducing isotropic tumbling motions of the spin-labeled molecules and, consequently, enhancing the ESR spectroscopic resolutions in the anisotropy of the local environment. These spectroscopic improvements made it possible for ESR techniques to study protein dynamics at room/physiological temperatures in the absence of viscosity agents that could possibly disrupt protein dynamical structure. However, a theoretical model for analyzing the ESR spectra of confined biomolecules has yet to be developed. To gain the benefits of the nano-confinement effects, it is urgently necessary to apply a more rigorous theoretical model of ESR lineshape theory to the analysis of the spectra collected under nano-confinement. This study incorporates the tether-in-a-cone (TIAC) model into the analysis of the inter-spin distance distribution of cw-ESR spectra. TIAC was previously developed by Eric J. Hustedt and applied to ESR spectra of a disordered system in a frozen solution state. The analysis was found to be very limited due to the poor cw-ESR resolution in the disordered system. In the TIAC model, a detailed molecular model describing the magnetic and molecular frames of each single nitroxide spin, the relative orientations of each spin pairs, and the magnitude of nitroxide side-chain disordering in terms of the cone width is taken into consideration in the spectral simulations. In this study, we follow the theoretical work of Eric J. Hustedt and develop a home-written Matlab program of the TIAC model calculation. In Chapter 1, we give a brief introduction concerning spin-label ESR techniques, the MSU mesoporous material, and the studied model peptide n3, which is a 26-residue-long polypeptide derived from prion protein. In Chapter 2, we give theoretical background and detailed descriptions as well as equation derivations of the TIAC model. In the first part of Chapter 3, a series of numerical model experiments are performed to demonstrate the validity of the program and the feasibility of the TIAC model for analyzing the spectra of spin-labeled biomolecules in nanochannels. In the second part of Chapter 3, a study of the real experimental data analysis using the TIAC model is presented. The TIAC program is utilized to extract the inter-spin distance distributions of the doubly labeled n3 polypeptides. A comparison of the TIAC results and the previous study based on point-to-point assumption is provided. With this sophisticated model, we demonstrate in both the studies of the model and experimental analyses that the use of the TIAC model is useful for revealing the relative orientational information that is essential to understanding the biomolecular conformations in the nanochannels. A concluding remark and summary of the improvements and perspective are given in Chapter 4.

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