利用生物物理的方法研究蜘蛛電壓調控毒蛋白與脂膜之交互作用

Abstract

Hanatoxin (HaTx) 是由蜘蛛分泌的神經毒性胜肽，可抑制鉀離子通道蛋白的功能，此胜肽會先與細胞膜磷脂質作用後再與膜內的鉀離子通道蛋白電壓感應片段作用來達到抑制的功能。由於深埋於膜內的電壓感應片段會隨著膜電位的變化藉由運動進而改變其所在位置，但是此類水溶性的胜肽如何改變電壓感應的機制仍不清楚。此外，由此類胜肽所造成的膜擾動機制亦尚未明瞭。因此，我們提出一系列的生物物理及生物化學的方法來了解HaTx和膜作用時可能的動態運動模式。首先，為了釐清HaTx與膜結合的細節，利用單層膜模擬雙層膜的外層，觀察到HaTx會進入至磷脂質的疏水長碳鏈中。再進一步了解有關膜的生物物理性質，也就是探究HaTx如何造成脂質雙層膜的擾動。在接近完全水合的狀態下，利用多層膜X光繞射來觀察膜厚度的變化。最後以中子反射來定性量測HaTx穿入膜的深度，來了解其穿入深度與電壓感應片段作用位置的相互關係。實驗證明HaTx可穿透膜表面而進入疏水長碳鏈 (從膜表面往內估算大約~9 Å處)，與先前研究的VSTx比較，其作用於膜表面的方式不同。兩者的差異在於穿入膜的深度不同，可藉由此深度來推估其對應的電壓感應片段位於膜內不同位置，進而解釋兩種鉀通道蛋白不同的抑制效應。此外，透過HaTx穿入膜深度的實驗數據進行分子模擬 (MD-simulations) 可以清楚顯示HaTx與膜作用時的適當結合位向，此與快速動力學(stopped-flow analysis)所提供的結果一致。綜合以上實驗結果，可以詳細描述HaTx與細胞膜的相互作用，進而瞭解並闡明鉀離子通道的抑制機制。

Hanatoxin (HaTx) from spider venom functions as an inhibitor of Kv2.1 channels, most likely by interacting with phospholipids prior to affecting the voltage-sensor (VS). However, how the water-soluble peptide modifies the gating remains poorly understood, as the voltage-sensor is deeply embedded within the bilayer, although it would change its position depending on the membrane potential. Furthermore, membrane perturbation induced by cysteine-rich peptides is a crucial biological phenomenon but scarcely investigated. As a result, we proposed a series of effective biophysical-chemical methodologies to illustrate the possible dynamic scenario of HaTx prior to binding and embedded within membranes. To clarify the binding details, Langmuir trough experiments were performed with phospholipid monolayers by mimicking the external leaflet of membrane bilayers, indicating the involvement of acyl chains in such interactions between HaTx and phospholipids. Getting a close step to understand the details of membrane biophysics, in other words, to see how HaTx interacts with phospholipid bilayers, we observe the toxin-induced perturbation on POPC/DOPG-membranes through measurements of the change in membrane thickness via Lamellar X-ray diffraction (LXD) on stacked planar bilayers in the near-fully hydrated state. Quantitatively, to see whether HaTx can indeed bind to the voltage-sensor, the depth at which HaTx penetrates into the POPC-membrane was measured with neutron reflectivity. Our current results successfully demonstrate that HaTx penetrates into the membrane hydrocarbon core (~9 Å from the membrane surface), not lying on the membrane-water interface as reported for another voltage-sensor toxin (VSTx). This difference in penetration depth suggests that the two toxins fix the voltage-sensors at different positions with respect to the membrane normal, thereby explaining their different inhibitory effects on the channels. In particular, results from MD-simulations constrained by our penetration data clearly demonstrate an appropriate orientation for HaTx to interact with the membrane, which is in line with the biochemical information derived from stopped-flow analysis through the delineation of the toxin-VS binding interface. To summarize all of current results, we can speculate the inhibitory mechanism of Kv channels through the detailed interactions between HaTx and membranes.