LT-IIb是大腸桿菌中的一種忌熱型腸毒素，具有AB5結構並導致腹瀉。第二型b型忌熱型腸毒素之 B 次單元 (LT-IIb-B5) 形成一個環形的五聚體，其下部區域會固定至細胞膜上的GD1a神經節苷脂，上部區域則會與類鐸受體2/1 (TLR2/TLR1) 結合，從而激活NF-ĸB的訊息傳遞路徑並活化先天性免疫反應。免疫系統的活化使得LT-IIb-B5有望成為重組次單元疫苗的粘膜佐劑。過去，LT-IIb-B5與Neu5Ac-nLT複合物的晶體結構被報導，揭示了特異性結合位點包含Trp92、Asn32、Asn31、Ile30、Thr14、Thr13和Arg12殘基。然而，LT-IIb-B5與GD1a的結合特徵仍然難以捉摸。為了深入了解LT-IIb-B5和GD1a-os的相互作用，我們表現並純化了經過氘化、碳13 和氮15同位素標定的 LT-IIb-B5，以獲得高品質的三維核磁共振的數據。迄今為止，我們完成了LT-IIb-B5骨架中大部分的氫、碳13和氮15化學位移的標定，這使我們能夠通過TALOS+預測LT-IIb-B5的二級結構。此外，還分析了每個殘基的氫/氘交換速率和骨架動態。LT-IIb-B5與GD1a-os的結合親和力是透過核磁共振滴定實驗以及其他生物物理實驗，如等溫滴定微量熱儀、生物膜干涉技術等進行的。儘管生物物理研究表明LT-IIb-B5和 GD1a-os 之間的交互作用非常微弱，化學位移的擾動說明了Ile49-Asp54的loop區域有著重要的作用。綜上所述，這項研究從溶液狀態的角度提供了一些有價值的訊息，有助於LT-IIb-B5相關的疫苗開發和應用。

Escherichia coli LT-IIb, one of heat-labile enterotoxins, features an AB5 structure and causes diarrhea. The B subunits of LT-IIb form a torus-shape pentamer in which the lower region anchors to GD1a ganglioside on the cell membrane and the upper region binds to TLR2/TLR1 which enables NF-ĸB pathway activation and boosts innate immune responses. Stimulation of the immune system makes LT-IIb-B5 a potential mucosal adjuvant for recombinant subunits vaccine. Previously, crystal structure of LT-IIb-B5 in complex with Neu5Ac-nLT was reported, revealing the specific binding sites contain Trp92, Asn32, Ile30, Asn31, Asn32, Thr14, Thr13 and Arg12 residues. However, the binding characteristics of LT-IIb-B5 with GD1a remains elusive. To further gain insights into the interactions of LT-IIb-B5 and GD1a-os, we have expressed and purified perdeuterated 2H, 13C, 15N-labelled LT-IIb-B5 for obtaining good quality of 3D NMR data. To date, we have assigned most of the LT-IIb-B5 backbone 1H, 13C, 15N resonances, which enables us to predict the secondary structure of LT-IIb-B5 by TALOS+. Furthermore, H/D exchange rates and backbone dynamics on each residue have been analyzed. Binding affinity of LT-IIb-B5 to GD1a-os was performed by NMR titration experiment as well as other biophysical experiments, such as ITC, BLI, etc. Even though biophysical studies reveal very weak interaction between LT-IIb-B5 and GD1a-os, mapping of chemical shift perturbations suggests that the loop region of Ile49-Asp54 plays an important role. Taken together, this research study has provided some valuable information from solution-state perspective which may be useful for the vaccine development related to LT-IIb-B5.

摘要 i
Abstract ii
Abbreviation iii
1. Introduction 1
2. Materials and methods 5
2.1 Expression and purification of LT-IIb-B5 5
2.2 Circular dichroism 7
2.3 Analytical ultracentrifuge 7
2.4 Coupling size-exclusion chromatography with multi-angle light scattering (SEC-MALS) 8
2.5 NMR studies 8
2.5.1 NMR experiments and assignments 8
2.5.2 The H/D exchange study 9
2.5.3 Relaxation data analysis 9
2.5.4 Titrated GD1a-os to LT-IIb-B5 10
2.6 Isothermal titration calorimetry (ITC) 11
2.7 Bio-layer interferometry (BLI) 11
3. Results 13
3.1 Purification and conformation of LT-IIb-B5 13
3.2 Resonance assignment and secondary structure prediction 14
3.3 Amide proton exchange rate 16
3.4 Backbone dynamics analysis 16
3.5 Mapping of interactions between LT-IIb-B5 and GD1a-os 17
3.6 Biophysical studies of GD1a-os to LT-IIb-B5 18
4. Discussion 20
5. References 23
6. Figures 27
Figure 1. Sequence alignment of type II heat labile enterotoxins. 27
Figure 2. Model showing the interactions of LT-IIb with Toll-like receptors (TLR2/TLR1) and GD1a that affect NF-𝛋B cell pathway [16]. 28
Figure 3. SDS-PAGE and gel filtration of LT-IIb-B5. 29
Figure 4. Circular dichroism spectra of LT-IIb-B5. 30
Figure 5. AUC and SEC-MALS showing LT-IIb-B5 forms a pentamer in pH 3. 31
Figure 6. Overlapping spectra of 13C, 15N-LT-IIb-B5 (blue) and 2H, 13C, 15N-LT-IIb-B5 (red). 32
Figure 7. 1H, 15N-TROSY HSQC spectrum of LT-IIb-B5 with assignment annotation. 33
Figure 8. The secondary structure of LT-IIb B5 predicted by TALOS+. 34
Figure 9.1. Comparison of the H/D amide proton exchange rate of LT-IIb-B5 in pH 3. 36
Figure 9.2. Mapping the H/D amide proton exchange rate to LT-IIb-B5 sequence and structure. 37
Figure 10. Backbone dynamics of LT-IIb-B5. 38
Figure 11.1. Titration experiments with GD1a-os. 39
Figure 11.2. LT-IIb-B5 interaction region with GD1a-os. 40
Figure 11.3. Perturbed residues of LT-IIb-B5 with GD1a-os and its critical binding sites reported previously [23]. 41
Figure 12. LT-IIb-B5 binding to GD1a-os measured by Isothermal calorimetry (ITC). 42
Figure 13. LT-IIb-B5 binding to GD1a-os measured by Bio-layer interferometry (BLI). 43
Table 1. Backbone resonance assignment of LT-IIb-B5. 44

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