生物分子聚集於空氣-水界面之實驗及模擬

本文屬最初階段的研究計劃，致力於開發一個新的流體力學的方法，以實現辨識分子生物學中某些重要的蛋白質結構（例如:薄膜蛋白質）。其主要目標是建立在空氣-水的化學官能界面中，增長二維蛋白質晶體的流體力學條件。這包括尋找最佳的流體力學條件，以大量且穩定的增長二維蛋白質晶體，即單分子層蛋白質晶體。獲得這種單分子層晶體將能利用X-射線繞射的技術進一步分析蛋白質結構。目前發展中的技術主要是利用靜置的水面上先覆蓋單分子層的脂質（在平衡狀態下產生的Langmuir膜，僅奈米大小的厚度）。溶解於亞相(subphase)水溶液中的蛋白質分子，在化學擴散的作用下，會選擇性的吸附（結合）於單分子層的脂質膜，形成脂質複合體（典型的脂質複合體，例如:生物血脂-抗生物素蛋白）。這種方式在界面中所形成蛋白晶體，受限於單分子層脂質膜在界面中的二維擴散，或多或少會形成類似於粉末狀結晶體。不規則的分子自我組裝所形成的粉末狀結晶體，不適合利用X射線繞射的技術來分析其蛋白質的結構。本論文的概念是藉由控制亞相水溶液的循環流動，讓： - 蛋白質分子加快吸附於亞相水溶液介面上的脂質，自我組裝為脂質複合體（脂質-蛋白質）， - 讓吸附於界面中的脂質複合體排列緊密， - 最終使二維分子的自我組裝更加容易，以形成排列整齊的二維蛋白質晶體。利用這樣的概念，我們建立實驗和理論的工具，以利於實現一個促進二維蛋白質晶體生長的過程。首先，我們使用脂肪酸（十五碳酸，pentadecanoic acid）模擬單分子層的脂質膜結構受到流動影響下的行為。要實現這樣的構想，實驗裝置設計為一環形渠道，由環形渠道底部的環形磁環旋轉而渠道兩側的垂直壁面固定不動，產生剪切性質的流動（或漩渦）。從流體力學的觀點來看，這樣的流動會使渠道的界面上所形成的單分子層的十五碳酸（或十五碳酸薄膜）同時受到兩種類型的剪應力（一個是渠道整體流動所造成的剪應力，另一個是界面上的流動所造成的剪應力）。我們研究十五碳酸單分子層同時承受這兩個剪應力時的行為。在十五碳酸薄膜中新的界觀的組織結構所形成的二維的共存相（即所謂的擴散液體相和壓縮液體相）仍被維持，即使其完全不在熱力學穩態平衡狀態下。利用Brewster角顯微鏡，測量界面中十五碳酸薄膜的壓縮液體相的分量分佈，我們已經證實，渠道底部的旋轉讓亞相水溶液流動而造成的離心力，會在亞相水溶液的界面（以大部分數據的觀察）產生一個沿徑向方向上向心力量的流動，它有利於壓縮液體相聚集在靠近渠道表面半徑較小處。在一個夠大的離心力作用下，Langmuir薄膜（十五碳酸薄膜）經由明顯的形態(或稱為圖案結構，這裡指的是擴散液體相和壓縮液體相兩相共存的分佈狀態)上的轉變，維持剪應力和線張力(類似一維表面張力)兩者之間的平衡。因此，剪切所導致的壓縮液體相的熔化，會生成新的圖案結構，類似一個二維驅散的小凝結液滴。論文最後我們建立一個分析前述環形渠道剪切流動的數學模型。渠道上方的液體表面覆蓋一層表面活性劑。假設流體流動為永久的，軸對稱且為潛變流（低Reynolds數，Stokes近似)。由於亞相水溶液深度和渠道外徑的比值夠小（淺流），所以能發展漸近匹配技術來求解。在旋轉的亞相液體中，核心流動可以從沿著側壁的邊界層區分出來。該模型能考慮到由離心力所造成的渠道表面徑向方向上分子向內(半徑較小處)聚集的影響。更具體地說，此數學模型藉由考慮在液體表面的突然轉變之動量平衡（Boussinesq-Scriven平衡），來分析徑向方向表面粘度的分層現象。

關鍵字

環型流動；剪切；界面劑； Langmuir單分子層；表面黏性； Stoke潤滑

並列摘要

This dissertation is the first stage of a research program devoted to the development of a new hydrodynamic process in which the object is to facilitate the recognition of certain important proteins structures (such as membrane protein) in the field of molecular biology. The major objective is to find hydrodynamic conditions which favor the growth of a two-dimensional (2-D) crystal of proteins at a chemically-functionnalized air-water interface. It includes researching the best hydrodynamic conditions for growing 2-D crystal protein sufficiently large and regular, e.g. a single (mono) crystal. Obtaining such a single (mono) crystal would helpful for X-ray diffraction technique to identify the primary structure of a protein rapidly. The state of the art today is based upon a water surface at rest, functionalized (covered) by a monolayer of lipids thus obtained for Langmuir film (nanometer thick) at equilibrium. Under the effect of chemical diffusion within water subphase, solubilize dproteins in the subphase adsorb to the lipids specifically designed to trap them (consider for instance the molecular complex [biotinylated lipid–avidin protein]). A protein crystal obtained at 2-D interface limited to its 2-D diffusion in the lipid monolayer is more like a crystalline powder. The irregularity of the molecular self-assembly in the crystalline powder is particularly ill-suited to the X-ray diffraction technique to identify its structure. The aim of this dissertation is to control a recirculating flow in the subphase to: - accelerate the capture of proteins to lipids presented in the liquid surface, - densification of 2-D complexes [lipid–protein] after adsorption, - ultimately, should logically lead to a 2-D single crystal assembly. This dissertation contributes to the experimental and modeling tools needed to develop a enhanced 2-D single crystal protein assembly. This dissertation contributes to the experimental and modeling tools needed to develop a enhanced 2-D single crystal protein assembly. A first part of this dissertation is devoted to the experimental set-up which is based upon an annular channel whose floor is put in rotation whilst its two vertical (cylindrical) side walls are maintained stationary. The channel is filled with a supporting subphase of acidified ultra-pure water. In order to confirm the feasibility of a flow-induced molecular densification, the user-friendly pentadecanoic acid (PDA) is chosen to mimic the response of a lipidic Langmuir monolayer when it is put out of equilibrium. A monolayer of PDA is therefore submitted to an annular shear flow. we have studied the behavior of a monolayer of pentadecanoic acid (PDA) simultaneously subject to two types of shear, one is in-plane shear, the other one is subphase shear valued at the surface. It is worthy to note that transition between liquid-expanded and liquid-condensed phases is conserved even in conditions far from thermodynamical equilibrium. Brewster angle microscopy (BAM) is used to image selectively the mesoscopic morphology of the subsequent two-phase PDA film. The area fraction of the condensed phase is carefully investigated after a permanent regime is established. The distribution of the area fraction demonstrates radially-inwards packing along the liquid surface which is induced by a centripetal surface flow originating from centrifugation of subphase along the rotating floor. For a growing level of centrifugation, a circular segregation front arises along the liquid surface. For a high enough level of centrifugation, the Langmuir film even experiences a strong morphological transition driven by a balance between surface shearing and reduced line tension. As a result, a shear-induced melting of the condensed phase generates a new patterning which can be described as a 2-D monodispersed matrix of tiny condensed droplets. The last part of this dissertation is devoted to modelling the previous annular shear flow. The liquid surface at the top of the channel is again supposed to be covered by a layer of chemically-functionnalised hydrophobic molecules. The flow is considered as permanent, axisymmetric and creeping. The ratio of the liquid depth to the outer radius of the channel is small enough (shallow flow) so that it is possible to develop a matched asymptotic technique. In the rotating subphase, a core flow is therefore distinguished from the boundary layers along side walls. The modeling includes the possibility to take into account the impact of the radially-inwards molecular packing induced by centrifugation of the underlying bulk. More particularly, radial stratification of surface viscosity is taken into account via the jump momentum balance at the liquid surface (Boussinesq-Scriven balance).

並列關鍵字

Annular flow ； shear ； surfactants ； Langmuir monolayer ； surface viscosity ； Stokes lubrication

參考文獻

[1] N. K. Adam. The properties and molecular structure of thin films. Part II.—

Condensed films. Proc. R. Soc. London, Ser. A, 101(712):452–472, 1922.

[2] A. W. Adamson and A. P. Gast. Physical chemistry of surfaces. John Wiley

LE-LC phase transitions in Langmuir monolayers of fatty acids. J. Phys.

and L. Zou. Domain relaxation in langmuir films. J. Fluid Mech., 571:191–219,

國際替代計量

生物分子聚集於空氣-水界面之實驗及模擬

全文下載

主題瀏覽