質子傳送-焦磷酸水解酶(簡稱H+-PPase; EC 3.6.1.1)是維持生物體內pH恆定的重要酵素。這個獨特的質子傳送酶藉由水解生物次級代謝產物－焦磷酸產生能量以驅動質子傳送，來維持細胞膜內外的酸鹼平衡。已知此酵素的焦磷酸水解功能單位為單元體，而質子傳送功能單位為同雙元體，其每個單體的酵素活化中心均由位在第五環圈的焦磷酸結合區段與第一酸性區段，以及位在第十五環圈的第二酸性區段和數個基要胺基酸所構成。本實驗即利用單分子螢光共振能量轉移技術，量測這些重要區段及胺基酸之間的分子間距離，並觀察此酵素與受質類似物及離子結合時所發生的之距離變化。其中位在同雙元體上兩個羧酸端的距離為49.3 ± 4.0 Å，而兩個胺基端之間的距離為67.2 ± 5.7 Å。雖然兩個焦磷酸結合區段的距離相對遙遠(70.8 ± 4.8 Å)，但是當質子傳送-焦磷酸水解酶與鉀離子及焦磷酸類似物結合後，兩個焦磷酸結合區段會變得更靠近彼此(56.6 ± 4.1 Å)。此外當酵素與受質類似物結合時亦會引起同雙元體上兩個第一酸性區段及兩個H622胺基酸的距離發生重要的改變，然而此現象在鉀離子與質子傳送-焦磷酸水解酶結合時並未發生。因此，本研究在同雙元體質子傳送-焦磷酸水解酶的基要區段、重要胺基酸以及酵素活性區段之間的距離量測上提供了重要的結構意義，並且提出一個酵素與受質結合機制的模式。

Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel enzyme supplies energy at expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit of a monomer suffices for enzymatic reaction of H+-PPase, while that for the translocation is homodimer. Its active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation, structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 Å and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), while binding of potassium and substrate analogue led them to closer proximity (56.6 ± 4.1 Å). Moreover, substrate analogue but not potassium elicits significantly distance variations between two Acidic I motifs and two H622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.

Abbreviations………………………………………………………………………..9
Introduction…………...…………………………………….……...………..……….10
Experimental Procedures....…………………...…………………………...………..13
CtH+-PPase DNA construction and mutagenesis…………..………….…………13
Microsome isolation and protein purification.....………….………......................13
CtH+-PPase activity assays……...………………….……………………………15
Atomic force microscopy………….…………………..……….…………..…….16
Fluorescence spectroscopy…...…………..……………………...……………….17
Single molecule imaging assays…………………………………………………18
TEM image analysis………………………………………………………..…….19
Results and Discussions………..………………………………………..……..…….22
Construction and characterization of CtH+-PPase mutants………........................22
Determination of inter-subunit distance for both termini and two active sites......25
Ligand effects on FRET efficiency between two termini and two active sites......30
References…………………...………………………………………………………..35
Table and Figures……………...…..……………………………...……….................42
Table I. FRET efficiencies (E*) and calculated distances (R) between dye pairs
in the absence and presence of ligands…................................................42
Figure 1. Topological model of CtH+-PPase.……………….……………...….....43
Figure 2. Heterologous expression and purification of CtH+-PPase…………......44
Figure 3. Enzymatic activities of CtH+-PPase.…………………………………..45
Figure 4. Expression and PPi hydrolyzing activities of CtH+-PPase mutants.…...46
Figure 5. PPi supported H+ translocation activities of CtH+-PPase mutants..........47
Figure 6. Coupling efficiencies of CtH+-PPase mutants.……………………...…48
Figure 7. smFRET determination of homodimeric CtH+-PPase.……………...…49
Figure 8. Histograms of FRET efficiencies.…………………………………..…50
Figure 9. TEM analysis of GNP-labeled homodimeric CtH+-PPase………….…51
Figure 10. K+ effects on the FRET efficiencies………………………………….52
Figure 11. IDP effects on the FRET efficiencies………………………………...53
Figure 12. K+/IDP effects on the FRET efficiencies………………………..……54
Figure 13. Substrate-mediated conformational change in the catalytic sites of
homodimeric CtH+-PPase…………..………………………………..55
Appendix………………………………………………………………………….…..56
Figure S1. Construction of CtH+-PPase.……………………………………..…..57
Figure S2. Determination of the optimal isopropyl β-D-thiogalactoside concentration for the expression of CtH+-PPase in E. coli…...………58
Figure S3. Time-dependence of CtH+-PPase gene expression in E. coli C43(DE3) at 37℃ with β-D-thiogalactoside treatment……………………….....59
Figure S4. Scheme of the components of TIRFM used for smFRET determination
……………………………………………………………………………………60
Figure S5. Schematic drawing for the determination of fluorescence anisotropy
..………………………………………………………………………………….61
Figure S6. Anisotropy of Alexa 488 and 647 at modified sites in putative active sites, N and C termini of CtH+-PPase………………………………..62
Figure S7. Sequence alignment of H+-PPases and sites of probes labeling in CtH+-PPase…..……………………………………………………….64
Figure S8. Determination of the optimal pH for CtH+-PPase……..……………..65
Figure S9. Kinetic analysis of wild type CtH+-PPase……………………………66
Figure S10. Histogram of purified CtH+-PPase height determined using the
AFM image…………………………………………………………67
Figure S11. Histogram of purified CtH+-PPase width determined using the
AFM image.………………………………………………………….68
Figure S12. Absorption and fluorescence emission spectra of Alexa Fluor 647
and Alexa Fluor 488 labeling at CtH+-PPase in pH7.2 buffer……….69
Figure S13. TEM analysis of monomaleimido GNP-labeled homodimeric CtH+-PPase………..………………………………………………….70
Figure S14. Histogram of the distances between GNP pairs determined by TEM
…….…………………………………………………………………71
Figure S15. Calculated distances between GNP pairs determined using TEM
……………………...………………………………………………...72
Figure S16. The proposed model based on smFRET measurement……………..73

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