外加電場環境下之分子馬達F0動態性質與細胞膜交互作用分析

Abstract

生物細胞的能量是從分解ATP分子而得到，ATP則可由F0F1 ATP合成酶來合成。存在於粒腺體內層細胞膜上的旋轉式分子馬達F0，對於驅動F1使之合成出ATP扮演很重要的角色。我們使用分子動力學來模擬b2-free F0 處在1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) 雙層磷脂質環境下的特性。藉由個別subunit helices之軸方向向量與POPC雙層磷脂質之垂直法向量的夾角(tilt angle)和個別N-terminal 與C-terminal helix的kink angle，我們可以了解經過分子動力學模擬處於雙層磷脂質的F0結構和原始結構的差異。藉由把F0 軌跡投影到principal component分析所得到之兩個主要的eigenvector所構成的平面後，可以很清楚地觀察到外加電場與其方向對F0軌跡所造成的影響。Principal component分析也成功地顯示F0在Asp61是否為電中性之影響下性質之差異性。這些結果是均方差異(RMSD)或旋轉半徑(radius of gyration)等分析無法得到的。不同的residues之間運動是否有相關性可由Pearson 相關系數分析來得到。外加電場會使residue之間的運動相關性降低。當Asp61轉變為電中性時，我們觀察到N-terminal helix中的Ala24和Ile28會旋轉30度。此旋轉運動主要是由於其N-terminal helix 和c環之第12個單位(c12)之間的靜電作用力所造成，此運動對subunit-c 之旋轉或許是一個必然的中間步驟。F0之兩個次單元(a-subunit與c-subunit)間的交互作用能量分析可以得到凡得瓦作用力是維持兩個次單元穩定結合的主要因素。觀察a-subunit內處於質子轉移路徑上之胺基酸間的氫鍵連接狀況，可觀察其氫鍵之穩定性。我們也發現到少許水分子會進入a-subunit內，此現象也和文獻相符合。我們也計算Asp61、Lys203和Glu219之intrinsic pKa 值，其均與實驗值十分接近。 要觀察F0與脂質(lipid)之間的交互作用，將脂質分成數層後分別計算各層脂質的deuterium order parameter (SCD)和其平面擴散係數。和單純雙層磷脂質比較的話，可觀察出整體脂質之SCD 並未明顯受到F0或外加電場的影響。然而，屬於不同層之脂質，其SCD值對於是否有受到F0或外加電場之影響則有顯著的差異。靠近F0蛋白質之第一層與在c-subunit內的脂質，其結構排列較為整齊。靠近F0之脂質或許對於蛋白質旋轉有潤滑效用。由各層脂質的平面擴散係數也可觀察到，F0會減低其擴散運動的程度。對於遠離F0的脂質則可看出其SCD與平面擴散係數都很接近於單純雙層磷脂質之性質。大體上說來，SCD與擴散係數呈現反比關係，換句話說，結構較整齊的脂質通常擴散速率較慢。我們的研究結果對於了解旋轉式分子馬達F0與周圍環境之脂質和外加電場的交互作用提供了分子層次的機制，同時也對於了解為何F0能高效率地轉換能量也提供了重要的相關資訊。

The membrane-bound component F0, which is one major component of the F0F1-ATP synthase, works as a rotary motor and plays the central role of driving the F1 component to transform chemi-osmotic energy into ATP synthesis. We have conducted molecular dynamics simulations of a b2-free F0 protein in the 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) lipid bilayer for tens of nanoseconds with two different protonation states of the cAsp61 residue at the interface of the a-c complex in the absence of electric fields and under electric fields of ±0.03 V/nm across this membrane. Principal component analysis (PCA) revealed that the all-protonated Asp61 cases indeed exhibit larger motion and the external electric fields strongly influence the dynamics of F0. Correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. When Asp61 were protonated, the Ala24 and Ile28 of the N-terminal helix rotated about its axis by 30°. This rotation induced by electrostatic interaction between N-terminal helix and C12 subunit may lead the conformational changes in the C-terminal helix which are important for the rotation of c ring. Van der Waals and Coulomb interactions between a- and c-subunit were calculated to confirm the former plays the dominant role in stabilizing two subunits which are hydrophobic. We also applied hydrogen bonds networks analysis to the residues on three individual proton translocation pathways to see whether the hydrogen bonds formed between these residues. Direct observation of the aqueous-accessible region within the a subunit is used to support there are water molecules in this region. The intrinsic pKa values of Asp61, Lys203 and Glu219 are supported by the experimental data. The deuterium order parameter (SCD) profile calculated by averaging all the lipids in the F0-bound bilayer was not very different from that of the pure bilayer system, which agrees with recent 2H solid-state NMR experiments (Kobayahi et al, Biophys. J., 94, 2008). However, by delineating the lipid properties according to their vicinity to F0, we found that the SCD profiles of different lipid shells are prominently different. Lipids close to F0 formed a more ordered structure. Similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. The lipids in the proximity of F0 exhibited very significantly reduced diffusional motion. The numerical value of SCD was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. These findings not only be useful to understand the dynamics of F0 related to the protonation state and electric fields, but may shed some light to the interactions between the motor F0 and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency.