蛋白質結構轉變的多尺度表現-從結構的非唯一性到蛋白質沉澱

Conformation transformations of proteins in multiple scales - from conformation non-uniqueness to aggregation

林俊羽

指導教授 ： 牟中瑜

國立清華大學/理學院/物理學系/博士(2014年)

https://doi.org/10.6843/NTHU.2014.00110

摘要

雖然一般相信根據Anfinsenm原理，蛋白質的結構會被它們的序列所唯一決定，但在多尺度下，某些複雜的自然態結構轉換表現出難以捉摸的現象。為了闡明多尺度下結構轉換的機制，我們從唯一結構這個問題去重新了解它，特別是從2DX4這個多肽鏈著手。2DX4包含18個胺基酸:INYWLAHAKAGYIVHWTA。基於一個我們全面校準過的第一原理粗粒化模型，我們建立了一個2DX4完整的自由能地形圖，揭示了2DX4天生是一個多結構共存的蛋白質。它的兩個自然態幾乎是簡併的:一個是螺旋結構，另一個是beta髮夾結構。雖然兩者之間存在著轉換的路徑，但是由於一個約10 kcal/mol的能障存在，兩個自然態之間的轉換並沒有很頻繁。這結果提供了一個重要的線索，小蛋白質的自然態可能不是唯一的所以結構轉換可能發生。我們因此把研究的範圍延伸到多肽鏈 A-beta(16-22)纖維化沉澱的模擬。我們展示了 A-beta(16-22)也有兩個自由能的最小值，一個是alpha-螺旋另一個是beta-髮夾彎結構。然而，因為它們兩個之間的能障很小，alpha與 beta之間的轉換變得很頻繁。結果就是，A-beta(16-22)並沒有固定的自然態。這樣的特性使的A-beta(16-22)表現出聚集沉澱和其他多重結構。由A-beta(16-22)的自由能地形圖可發現，它們的雙體乃至於六聚體的自由能完全與單體的自由能形式無關。反平行的 beta-摺板是這些多聚體的最低能量態，同時有幾個暫存態但是並不穩定。為了能模擬大尺度的沉澱，我們設計了一個盒子中間裝了一個結核的晶種讓其他單體與其反應。使用這個新穎的設計，我們能夠模擬不同濃度下A-beta(16-22)的沉澱行為。在低濃度下，模擬顯示了A-beta(16-22)類似奈米線的成長模式，纖維的成長是由擴散速率決定。然在高濃度下，系統傾向於非結晶且有多個最低能量態。我們的分析指出在低濃度下，沉澱的行為特徵可歸類為古典成核理論，包含遲滯期，臨界核，以及有序纖維化；而它們的成長路徑與濃度相依。這些結果讓人更明白蛋白質沉澱的多重態是與生俱來，且為了解蛋白質自組裝的沉澱路徑提供了重要線索。

關鍵字

蛋白質摺疊 ； 沉澱 ； 阿茲海默症 ； 自由能

並列摘要

Although it has been generally believed as the Anfinsen's dogma that protein structures are uniquely determined by their amino acid sequences, complex conformational transformations of native structures that occur in multiscales makes Anfinsen's dogma elusive. To illuminate mechanism behind conformational transformations in
multiscales, we start by revisiting the uniqueness problem of protein native structures by investigating the peptide 2DX4, comprising 18 amino acids: INYWLAHAKAGYIVHWTA. Based on an ab initio coarse-grained model that are comprehensively calibrated, we construct the complete free energy of 2DX4 and demonstrate that 2DX4 is an inherently multi-conformation peptide with two nearly degenerate native structures: one is a helix structure, while the other is a hairpin-like structure.
Although there exist pathways connecting two degenerate native structures, conformation switch between two native structures does not occur often due to the existence of an energy barrier of the order 10kcal/mol between them. These results provide important clues that the native structures for small proteins may not be unique so that conformation transformations may occur. Our investigation is then extended to simulate fibril aggregation of peptide A-beta(16-22). It is shown that A-beta(16-22) also possesses two minimum with alpha-helix and beta-hairpin structures. However, due to small energy barrier between alpha and beta structures, transformations
between these structures occur frequently. As a result, A-beta(16-22) does not exhibit a fixed native structure. This feature enables the aggregation and manifestation of multi-conformations for A-beta(16-22). The energy landscapes of A-beta(16-22) from dimer to hexamer is found to be completely uncorrelated with that of a single A-beta(16-22). Antiparallel beta sheets are found to be the ground state, while several intermediates are minor unstable minima. To simulate large scale aggregation, a box container with a seed in which other monomers start to interact with the seed is implemented. Using this new simulation scheme, we are able to simulate aggregations of A-beta(16-22) in different concentrations. It is shown that similar to the growth of nanowires, when the density of A-beta(16-22) is low. The aggregation is dominated by diffusion limited aggregation, while for high densities, the system tends to be amorphous with multiple ground states. Our analyses indicate that in the low density region, the aggregation can be characterized by classical nucleation theory, including the lag phase, critical nucleation, fibrilization and their pathway dependence on the concentration. These results shed light on the multi-formational nature of aggregated proteins and provide important clues to understand aggregations pathways for amyloid assemblies.

並列關鍵字

Protein folding ； aggregation ； amyloid beta ； energy landscape

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