CSαβ模組由一個α-螺旋以及一個反平行的β-摺疊組成，並由三或四對雙硫鍵連接，形成非常穩定的結構，這使得CSαβ模組成為一個很適合研究蛋白質工程學的支架。由於結構的相似性，我們用綠豆防禦蛋白VrD1作為含有CSαβ結構的模板，利用點突變技術去改造出一個蛋白質，不僅具有原本的生物活性，還有類似蠍毒蛋白抑制離子通道功能。有一類蠍毒蛋白 (α-KTxs)具有一段活性多肽K-C4-X-N 可以專一性地去抑制電壓門控鉀離子通道(Kv1)。根據序列比對分析，我們將VrD1對應到的胺基酸(N32C33K34G35)突變成α-KTxs活性肽鏈K-C4-X-N，也建構出VrD1-E8Q, VrD1-T39K和VrD1-Y41K，使VrD1與離子通道的作用位置能帶更多正電。我們測試這些蛋白的電生理活性與抑制麵包蟲α-澱粉水解酶的功能，以探討重組VrD1的突變的胺基酸對鉀離子通道抑制功能與VrD1原本的抗α-澱粉水解酶活性之間的關係。
實驗結果顯示單點的賴氨酸替換無法提升抑制電壓門控鉀離子通道的能力，且在Loop 3位置上或是附近的突變 (G35N、T39K和Y41K)會導致VrD1抑制TMA的能力下降。另一方面，α-KTxs的活性肽鏈K-C-X-N可提升VrD1抑制特定Kv1通道的功能且不影響原本阻斷TMA的活性。在此我們製造出了雙功能的重組蛋白質。然而，僅使用點突變不足以使一個非毒素的植物防禦素成為有效的抑制離子通道毒蛋白，這顯示出CSαβ模組的最佳分子設計條件還需要更多的研究。根據結構對接分析，序列的N端區域看似會在VrD1和Kv1通道作用時形成阻礙，這個發現可以做為未來研究非毒素CSαβ模組成為離子通道阻斷劑的一個可行的分子設計方向。

Cysteine-stabilized αβ (CSαβ) motif consists an α-helix and an antiparallel β-sheet, connected by three or four disulfide bonds which provide high stablity and make this motif as a suitable framework for molecular engineering. Due to structural similarity, we utilized Vigna radiata plant defensin 1 protein (VrD1) as a template containing CSαβ motif to engineer a modified protein bearing not only its original function but also the channel blocking activity like scorpion toxin by point mutagenesis method in this study. Sequence K-C4-X-N is the toxin signature of α-KTxs scorpion toxins, which specifically affect voltage-gated potassium (Kv1) channel. According to sequence alignment, we mutated the corresponding positions (N32C33K34G35) in VrD1 to the toxin signature (K-C4-X-N) of α-KTxs, and also constructed mutants VrD1-E8Q, VrD1-T39K, and VrD1-Y41K to increase positive charge on the binding interface to channel. Both the electrophysiological recording and Tenebrio molitor larvae α-amylase (TMA) inhibition assay were conducted to investigate the function and characteristics of modified VrD1 proteins.
The results showed that Lysine substitution could not increase hKv channel blocking ability of VrD1, and the mutations on or near Loop 3 of VrD1 (G35N, T39K and Y41K) reduced the TMA inhibition. On the contray, VrD1 bearing toxin signature K-C-X-N increased the inhibition to sepecific Kv1 channels and retain the TMA inhibition. Here, we created a dual functional protein; however, the ability of using point mutation to develope nontoxic plant defensin into an efficient ion channel blocking toxin was limited. According to docking analysis, the N-terminal region seemed to be the obstacle in the interaction between VrD1 and Kv1 channel. This provided a feasible direction for molecular design of nontoxic CSαβ motif into the potent ion channel blocker.

Chapter I Introduction 1
1.1 Protein engineering 1
1.2 Cysteine-stabilized αβ motif 1
1.3 Scorpion K+ channel toxins α-KTx 2
1.4 Plant defensin and Vigna radiata plant defensin 1 protein (VrD1) 3
1.5 Aim of this thesis 4
Chapter II Materials and Methods 5
2.1 Construction of site-directed mutated recombinant VrD1 (rVrD1) 5
2.2 Expression and purification of rVrD1 6
2.3 Protein concentration determination (BCA protein assay) 7
2.4 Protein identification 7
2.5 Sulfhydryl assay (Ellman’s test) 8
2.6 Secondary and tertiary structure analysis 8
2.7 Purification of Tenebrio molitor larvae α-amylase 9
2.8 TMA activity assay and inhibitory function assay of rVrD1 10
2.9 Electrophysiology study (patch clamp whole cell recording) 11
Chapter III Results and Discussion 12
3.1 Construction of point mutated rVrD1 12
3.2 Purification and identification of rVrD1 12
3.3 Characterization of rVrD1 14
3.4 TMA inhibition assay of rVrD1 15
3.5 Electrophysiological Recordings 17
3.6 Molecular modeling and docking simulation 19
Chapter IV Conclusion 22
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