我們研究了極端嗜熱古菌Pyrococcus horikoshii 的嗜熱性酰基磷酸酶acylphosphatase (PhAcP) ，以及與它同源的人類嗜溫性酶(HuCTAcP) 的熱穩定性。我們發現PhAcP的熱穩定性之所以比HuCTAcP高出很多，是由於熔融溫度的焓變值的增加以及變性熱容量的減少。研究蛋白質熱穩定性的其中一個推動力，是運用我們的知識去製造耐高溫的酶，這對工業和生物技術非常重要。通過交換 PhAcP的嗜熱核和 HuCTAcP的嗜溫核以及研究變種的熱穩定性，我們認為蛋白表面是改善熱穩定性工程的首選地區。嗜熱和嗜溫蛋白質之間的主要區別，在於嗜熱蛋白質有更多的表面鹽橋。為了探討表面鹽橋對蛋白熱穩定性的貢獻，我們採用雙突變循環，量化嗜熱蛋白T.celer L30e一表面鹽橋的相互作用能。我們的結果顯示，表面鹽橋對蛋白質穩定性的貢獻是獨立於溫度變化的。此外，表面鹽橋對蛋白質變性熱容量的減少起一定作用。 / We characterized the thermodynamic properties of thermophilic acylphosphatase from Pyrococcus horikoshii (PhAcP) and its mesophilic homologue from human (HuCTAcP) and found that the much higher thermostability of PhAcP was the result of increased enthalpy change at melting temperature and decreased heat capacity change of unfolding. One incentive to study protein thermostability is to apply our knowledge to engineer thermostable enzyme which is of great industrial and biotechnological importance. Through swapping the core of thermophilic PhAcP and mesophilic HuCTAcP and characterizing the thermostability of the resulting variants, we concluded that surface is a preferred region for thermostability engineering. The key difference between thermophilic and mesophilic proteins lies in the surface on which thermophilic proteins have more salt-bridges. To investigate the contribution of surface salt-bridge to protein thermostability, we employed double-mutant cycle to quantify the pair-wise interaction energy of a surface salt-bridge in thermophilic T.celer L30e. Our results showed that surface salt-bridge had a temperature independent contribution to the protein stability and plays a role in the reduction of the heat capacity change of unfolding. / Detailed summary in vernacular field only. / Yu, Tsz Ha. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 89-93). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Content --- p.iv / List of Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.ix / Chapter Chapter 1: --- General introduction --- p.1 / Chapter 1.1 --- Definition of protein stability --- p.1 / Chapter 1.2 --- Contribution to thermostability from the protein core --- p.2 / Chapter 1.2.1 --- Definition of hydrophobic effect --- p.2 / Chapter 1.2.2 --- Why the hydrophobic effect has been recognized as the major driving force for protein folding? --- p.2 / Chapter 1.3 --- Contribution to thermostability from the protein surface --- p.6 / Chapter 1.3.1 --- Electrostatic interactions --- p.7 / Chapter 1.3.2 --- Exposed hydrogen bonds and helix propensity --- p.10 / Chapter 1.3.3 --- Surface loop --- p.11 / Chapter 1.4 --- Protein stability curve --- p.13 / Chapter 1.5 --- The incentive to study protein thermostability --- p.17 / Chapter Chapter 2: --- Materials and Methods --- p.18 / Chapter 2.1 --- Generation of DNA clones --- p.18 / Chapter 2.2 --- Plasmid transformation to competent E. coli strain --- p.18 / Chapter 2.3 --- Expression of recombinant proteins --- p.19 / Chapter 2.3.1 --- T. celer L30e --- p.19 / Chapter 2.3.2 --- Acylphosphatase --- p.20 / Chapter 2.4 --- Protein extraction from E. coli by sonication --- p.20 / Chapter 2.5 --- Protein purification --- p.20 / Chapter 2.5.1 --- T. celer L30e --- p.20 / Chapter 2.5.2 --- Acylphosphatase --- p.22 / Chapter 2.6 --- Circular dichroism experiment --- p.22 / Chapter 2.6.1 --- Thermal denaturation --- p.22 / Chapter 2.6.2 --- Denaturant-induced denaturation --- p.23 / Chapter 2.7 --- Differential scanning calorimetry --- p.24 / Chapter 2.8 --- Enzymatic assay of AcPs using benzoyl phosphate as substrate --- p.25 / Chapter 2.9 --- Crystallization and crystal structure refinement --- p.26 / Chapter Chapter 3: --- Thermodynamic characterization of thermophilic acylphosphatase from Pyrococcus horikoshii and its mesophilic homologue from human --- p.27 / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Result --- p.31 / Chapter 3.2.1 --- PhAcP has a higher thermostability than HuCTAcP --- p.31 / Chapter 3.2.2 --- PhAcP has an upshifted and broadened PSC compared with the PSC of HuCTAcP --- p.33 / Chapter 3.2.3 --- PhAcP has a highly enhanced ΔH[subscript m] and slightly reduced ΔC[subscript p]. --- p.37 / Chapter 3.3 --- Discussion --- p.41 / Chapter 3.3.1 --- Thermophilic AcPs harness enhanced ΔH[subscript m] and reduced ΔC[subscript p] to attain a higher thermostability. --- p.41 / Chapter 3.3.2 --- Possible structural differences between PhAcP and HuCTAcP that lead to the higher thermostability of PhAcP. --- p.42 / Chapter Chapter 4: --- Protein surface is a preferred region for thermostability engineering --- p.47 / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Results --- p.51 / Chapter 4.2.1 --- Construction of the chimera with Thermophilic Surface and Mesophilic Core (T[subscript surf]M[subscript core]), and the chimera with Mesophilic Surface and Thermophilic Core (M[subscript surf]T[subscript core]). --- p.51 / Chapter 4.2.2 --- The crystal structures of the chimera T[subscript surf]M[subscript core] and M[subscriptsurf]T[subscript core] reveal that anticipated interactions are engineered. --- p.54 / Chapter 4.2.3 --- Characterization of the thermodynamic stabilities of the chimeras at different temperatures --- p.56 / Chapter 4.3 --- Discussion --- p.59 / Chapter 4.3.1 --- Engineering a thermophilic surface onto a mesophilic protein enhances thermostability --- p.59 / Chapter 4.3.2 --- Concluding remarks --- p.64 / Chapter 4.4 --- Supplementary Tables --- p.64 / Chapter Chapter 5: --- Stabilizing surface salt-bridge enhances protein thermostability by upshifting the protein stability curve --- p.68 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.2 --- Results --- p.70 / Chapter 5.2.1 --- Design of variants --- p.70 / Chapter 5.2.2 --- Determination of the pair-wise interaction energy of K46 and E62 by double-mutant cycles --- p.72 / Chapter 5.2.3 --- Surface salt-bridge K46/E62 is stabilizing and its interaction energy is insensitive to temperature changes --- p.75 / Chapter 5.2.4 --- Stabilizing salt-bridge K46/E62 reduces ΔC[subscript p] and upshifts protein stability curve --- p.77 / Chapter 5.3 --- Discussion --- p.80 / Chapter 5.3.1 --- Stabilization effect brought by surface salt-bridge is insensitive to temperature change --- p.80 / Chapter 5.3.2 --- The pair-wise interaction energy of K46-E62 determined by DMC reflects their electrostatic interaction --- p.80 / Chapter 5.3.3 --- Surface salt-bridge contributes to the reduction of ΔC[subscript p] in thermophilic proteins --- p.81 / Chapter 5.3.4 --- Reduced ΔC[subscript p] upshifts and broadens the PSC resulting a higher T[subscript m] --- p.83 / Chapter 5.4 --- Supplementary Figures and Tables --- p.85 / Chapter Appendix --- List of Publications --- p.88 / References --- p.89