乙醯膽鹼（acetylcholine）所調控之下游訊息與影響神經傳導之功能主要透過兩種不同類型的乙醯膽鹼受體（acetylcholine receptor）進行傳遞，分別是nicotinic與muscarinic乙醯膽鹼受體，兩者皆屬於離子閘門通道家族的成員。nicotinic乙醯膽鹼受體是由五個不同次單元（subunit）所組成的五倍體穿膜蛋白，截至目前為止，已經有十七個次單元（alpha1-10, beta1-4, gramma,delta and epslion）在哺乳細胞中被發現且組成各種不同類型之nicotinic乙醯膽鹼受體，在人類細胞中已經有十八種不同次類型（subtype）已被發現；而muscarinic乙醯膽鹼受體是單一個次單元之穿膜蛋白，包含了一個胞外區塊、七個穿膜區塊與一個胞內區塊，截至目前則有五種不同次類型被發現。研究指出，乙醯膽鹼受體對於細胞生理狀態的影響極為顯著，因此與乙醯膽鹼受體相關之疾病漸受重視，例如重症肌無力（myasthenia gravis）這類自體免疫疾病已證實與nicotinic乙醯膽鹼受體有直接關聯性，而與其相關結構與功能的研究也漸趨廣泛，近年來更是藥物設計的重點之一。α神經毒蛋白與毒菌鹼毒素蛋白分別能專一性地與不同類型之nicotinic以及muscarinic乙醯膽鹼受體結合而抑制其功能。Erabutoxin-b（EBTb）屬於短鍊α神經毒蛋白成員之一，由海蛇Laticauda semifasciata的蛇毒純化得到；muscarinic toxin 2（MT2）則是毒菌鹼毒素蛋白的一員，由綠曼巴蛇（Green mamba）Dendroaspis angustuceps蛇毒所純化得到。由於EBTb與MT2皆具有類似的three-fingered fold：由四對高保留性的雙硫鍵構成中央疏水性核心以及兩個反向β薄板組成三維結構，但其卻能辨識不同的乙醯膽鹼受體，為了探討EBTb與MT2在功能、結構與動力學上之關聯性及如何辨識不同的乙醯膽鹼受體，我們利用Pichia pastoris系統表現EBTb與MT2，再利用鎳離子螯和親和性液相系統與逆向高效能液相系統純化重組EBTb與MT2，產率分別為15±5 mg/L與5±3 mg/L；質譜分析鑑定也確定重組EBTb與MT2具有正確分子量，而其分子量理論值已扣掉形成四對雙硫鍵理應扣掉的八個氫原子分子量之數值，暗示著重組EBTb與MT2皆有四對雙硫鍵形成。利用NMR檢視重組EBTb與MT2初步確定具有正確的構形（folding），進一步利用NMR分析其立體三維結構，證實利用Pichia pastoris系統表現的重組EBTb如同自然界的EBTb一樣，呈現three-fingered fold。根據動力學分析，重組EBTb在loop II、loop III與C端具有介於皮秒至奈秒、毫秒至微秒的內部運動性質，然而loop I不僅活動性低於上述這三個區域，相較於β-sheet區域更緊密固定，其原因可能是loop I有分子間氫健形成的緣故。除此之外，蛋白整體、loop I、loop II、loop III與C端之平均S2值分別是0.857、0.880、0.769、0.807與0.808，由各區域平均S2值的比較可以印證loop II、loop III與C端具有較高活動性。而靠近loop 尖端區域的His6、Ser9、Thr13（loop I），Cys17、Gly20（連接loop I與loop II的β-turn），His26、Lys27（loop II），Gly49、Leu52（loop III）具有明顯慢速構形變化現象，此結果提供了loop區域的確是EBTb參與肌肉型nicotinic乙醯膽鹼受體結合主要區域的重要證據之一。比較EBTb、toxin-α、dendroapsin（Den）與cardiotoxin II（CTXII）的動力學性質，loop II與loop III在這四個三指毒素蛋白確實呈現較高活動性，loop I除了CTXII之外都是較緊密固定，而EBTb與CTXII相較於toxin-α與Den則擁有活動性較高的C端，因此C端對於EBTb與CTXII在功能上的角色可能是穩定整體結構，增進與其目標蛋白之結合親和力，但此假設仍需進一步探討。比較EBTb在功能、結構與動力學的結果，可以了解loop II與loop III尖端區域所呈現的快速運動對於參與肌肉型nicotinic乙醯膽鹼受體是不可或缺的，但慢速構形變化也可能扮演重要角色，例如Lys27；loop I上胺基酸側鍊的位向、動力學性質與官能基則是loop I參與受體結合的重要因素，例如Ser8的側鍊；而C端具有明顯的慢速構形變化，推測C端可能藉著與loop I、loop II交互作用而增進nAChR結合親和力。綜合上述，藉由比較不同三指毒素蛋白之動力學性質、結構與功能，可以證實loop區域的確是參與其目標蛋白的重要區域。

Acetylcholine signals through two types of unrelated membrane receptors referred to as nicotinic (nAChR) and muscarinic (mAChR) acetylcholine receptors. Both nAChRs and mAChRs belong to members of the ligand-gated ion channel superfamily. nAChRs are the assembly of five subunits and the combinatorial assembly of 17 subunits, including alpha1-10, beta1-4, gramma, delta and epslion, gives rise to diverse receptor subtypes with distinct structures and functions in nAChRs. In contrast, mAChRs are single subunit protein with seven transmembrance-spanning regions and a large cytoplasmic domain. Both nAChRs and mAChRs are involved in many important biological processes, and their malfunctioning are the cause or consequence of various diseases such as Myasthenia gravis. They make AChRs attractive therapeutic targets in pharmaceutical industry. alpha-Neurotoxins and muscarinic toxins obtained from snake venoms can potently block diverse nAChRs and mAChRs, respectively. Erabutoxin b (EBTb) is a short chain α neurotoxin isolated from sea snake Laticauda semifasciata and is a potent inhibitor of muscular type nAChR. Muscarinic toxin 2 (MT2) is isolated from Green mamba Dendroaspis Angusticeps and is a potent agonist of M1 mAChR. Both EBTb and MT2 are three-fingered toxins and possess a similar architecture with unrelated biological functions. They share a common structure consisting of a beta-sheet core that tightly cross-linked by four conserved disulfide bonds. To study function, structure, and dynamics relationships of EBTb and MT2, we expressed EBTb and MT2 in Pichia pastoris. We purified them to apparent homogeneity using Ni2+-chelating chromatography and reverse-phase HPLC. The yields of unlabeled and 15N-labeled EBTb and MT2 produced in Pichia pastoris were 15±5 mg/L and 5±3 mg/L, respectively. Mass analysis showed that the experimental molecular weight of EBTb and MT2 produced in Pichia pastoris deviated less than 1 Da when compared with theoretical value. The theoretical value was calculated by assuming all cysteines formed disulfide bonds, indicating the formation of four disulfide bonds in EBTb and MT2. We also used NMR spectroscopy to determine the folding and dynamical properties of EBTb and MT2. NMR analysis showed that EBTb expressed in Pichia pastoris exhibited three finger fold as native protein. Dynamical studies demonstrated that EBTb exhibited flexibility on the loop regions of motions on multiple timescales. For example, the tips of the loop I (Ser8), loop II (Glu31, Phe32, Arg33, and Gly34) and loop III (Thr45, Val46, and Lys47) in EBTb exhibited flexible motions on the ps/ns timescale. The order parameter S2 values of loop II, loop III and C-terminal are 0.769, 0.807, and 0.808, which were lower than the overall average S2 value of 0.857. We also found that the residues His6, Ser9, Thr13, Cys17, Gly20, His26, Lys27 and Leu52, which are located nearby the tips of loop region of EBTb, exhibited large amplitude motion on the us/ms timescale. Comparison of the dynamical properties of EBTb with other three-fingered toxins, such as dendroaspin (Den), toxin α, and cardiotoxin II (CTXII), showed that their loop II and loop III have higher flexibility than other regions. The loop I of these toxins are all rigid except CTX II; however, the C terminal region of EBTb and CTXII were more flexible than those of Den and toxin α. Our results suggest that the C-terminal region of EBTb may stabilize the loop I and loop II and enhance its binding to muscular type nAChR. In conclusion, three loops and the terminal regions might allow functional residues of three-fingered toxins to adopt a variety of conformational and dynamical arrangements to recognize their targets.

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