The Dynamics and Kinetics of Proton Related Biological Processes Público
Ban-Seok Jeong, Samuel (Summer 2018)
Abstract
The proton plays special roles in biological systems because of its unique characteristics. Due to its minuscule size and exorbitantly high charge density, the proton is extremely reactive thermodynamically as well as kinetically. Because a proton can be transferred by the Grotthuss mechanism, its diffusion rate is fast. Since water molecules can easily accept or donate a proton, protons are readily available in an aqueous environment. Therefore, nature has employed protons for many biological processes and energy storage as a proton gradient is the central pillar of bioenergetics. The structure of proteins as well as the protein-protein interactions are modulated by protons. Most enzymes show highly pH-sensitive activity profiles. One good example is the viral infection cycle of influenza A virus. After endocytosis, the low pH within the lysosome activates the viral membrane protein, M2 proton channel. M2 conducts protons unidirectionally into the viral lumen with high selectivity and is activated at acidic pH, which has been of great interest for drug development. In this dissertation, using a laser-induced fast pH-jump, the fluorescence change of Trp41 of M2 transmembrane domain (M2TM) was measured. A double exponential decay was observed, where the fast phase was ascribed to protonation kinetics (~2×1010 M-1s-1), while the slow phase was attributed to the subsequent conformation change (~4×103 M-1s-1). The conformation change of M2 was further confirmed by FRET studies, demonstrating that M2TM makes a transition toward a more ‘open’ structure at acidic pH. The second half of this dissertation examined excited state intramolecular proton transfer dyes and their applications as hydrophilic, long-lived reversible photo-acids and local hydration level sensors. A previously reported hydrophobic long-lived reversible photo-acid was derivatized to be more hydrophilic by adding a sulfonate group. Although synthesis was successful, the sulfonation decreased the pKa of naphthol, preventing intramolecular proton transfer and not allowing it to be long-lived. We also studied 2,7-DiazaTrp which was known to show distinct 500 nm fluorescence in the presence of water, making it a good local hydration sensor. The 2,7-DizaTrp mutant of mastoparan X showed how it interacts with a membrane by probing local hydration level change.
Table of Contents
Chapter 1: Introduction -------------------------------- ------------------------------------- 1
1.1 The Unique Characteristics of Proton -------------------------------- -------------------- 1
1.2 The Roles of Proton in Biochemical System -------------------------------- -------------- 5
1.3 The pH jump technique as a Tool to Study Proton Related Processes --------------------- 8
1.4 Influenza A M2 Proton Channel as a Model System -------------------------------------- 11
1.5 Excited-State Intramolecular Proton Transfer (ESIPT) ----------------------------------- 13
1.6 Dissertation Outline -------------------------------- ------------------------------- ---- 15
1.7 Reference -------------------------------- ---------------------------------------------- 17
Chapter 2: Proton Transport Mechanism of M2 Proton Channel Studied by Laser Induced pH-jump ------ 28
2.1 Abstract -------------------------------- -------------------------------- -------------- 28
2.2 Introduction -------------------------------- -------------------------------- ---------- 29
2.3 Experimental Section -------------------------------- -------------------------------- -- 33
2.4 Results and Discussion -------------------------------- -------------------------------- 36
2.5 Conclusions -------------------------------- -------------------------------- ----------- 50
2.6 Appendix -------------------------------- -------------------------------- ------------- 51
2.7 References -------------------------------- ------------------------------ -------------- 61
Chapter 3: pH Dependent Conformational Change of Influenza A M2 Proton Channel Revealed by FRET ---- 67
3.1 Abstract -------------------------------- -------------------------------- -------------- 67
3.2 Introduction -------------------------------- ----------------------------- ------------- 67
3.3 Experimental Section -------------------------------- -------------------------------- -- 73
3.4 Results -------------------------------- -------------------------------- --------------- 75
3.5 Discussion ------------------------------ -------------------------------- -------------- 79
3.6 Conclusions -------------------------------- -------------------------------- -----------84
3.7 Appendix -------------------------------- -------------------------------- ------------- 86
3.8 References -------------------------------- -------------------------------- ----------- 93
Chapter 4: Synthesis and Characterization of Water-soluble Long-lived Reversible Photoacid and Its Application to pH-jump Experiment ..100
4.1 Abstract ---------------------------- -------------------------------- ---------------- 100
4.2 Introduction -------------------------------- -------------------------------- ---------100
4.3 Experimental Section -------------------------------- -------------------------------- 104
4.4 Results and Discussions -------------------------------- ----------------------------- 106
4.5 Conclusions -------------------------------- -------------------------------- -------- 109
4.6 Appendix -------------------------------- -------------------------------- ----------- 111
4.7 References -------------------------------- -------------------------------- --------- 114
Chapter 5: Investigation of Interaction between Mastoparan X and Membrane by Using 2,7-DiazaTrp as a Local Hydration Level Sensor .. 117
5.1 Abstract -------------------------------- -------------------------------- --------------117
5.2 Introduction -------------------------------- -------------------------------- -------- 117
5.3 Experimental Section -------------------------------- -------------------------------- 121
5.4 Results and Discussions -------------------------------- ----------------------------- 123
5.5 Conclusions -------------------------------- -------------------------------- ---------131
5.6 Appendix -------------------------------- -------------------------------- ----------- 133
5.7 References -------------------------------- -------------------------------- --------- 135
Chapter 6: Conclusion -------------------------------- -------------------------------- -- 140
5.1 Summary -------------------------------- -------------------------------- ------------140
5.2 Perspective -------------------------------- -------------------------------- ----------141
5.3 References -------------------------------- -------------------------------- ---------- 146
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