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

Thesis (M.S. in biochemistry)--Washington State University, December 2009. / Title from PDF title page (viewed on Jan. 20, 2010). "School of Molecular Biosciences." Includes bibliographical references.

Thesis (Ph.D.)--Tufts University, 2002. / Adviser: Eliana De Bernardez Clark. Submitted to the Dept. of Chemical Engineering. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser. Med sammanfattning på svenska.

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2007. / Includes bibliographical references. Also available on the Internet.

It is vital for the survival of an organism that it can repair damage to its DNA. Exogenous and endogenous sources of damage are dealt with by a variety of repair pathways that have evolved to repair specific types of damage. Organisms in the archaeal domain, the third domain of life, contain homologues of many of the eukaryotic repair proteins, however little is known about how damage is detected in the archaeal domain. Microarray studies in the archaeal species Sulfolobus solfataricus determined a number of genes whose expression was effected by UV radiation (work by Dr D Götz). The change in expression of nine of these genes was confirmed by RT real time PCR. The expression of these genes was then investigated after exposure to different damaging agents, Mitomycin C, Methyl methane sulfonate, Phleomycin and Hydrogen peroxide. The expression of two genes, transcription factor tfb-3 and cell division control gene cdc6-2, was up regulated in all damage conditions. There was a huge induction of the dps-like gene (sso2079) after hydrogen peroxide damage. Transcription from this genes promoter was shown to be strong in vitro (work by Dr S Paytubi) suggesting a repressor was controlling the gene in vivo. A palindromic repeat in the promoter of the dps-like gene was used to ‘fish’ for a transcriptional repressor and the Sso2273 protein, a homologue of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheria, was identified as a possible repressor. Sso2273 was expressed and purified, and its crystal structure solved, its paralogue, Sso0669, was also expressed and purified. Electrophoretic mobility shift assays showed that the Sso2273 protein does not bind DNA, and had no effect on transcription from any promoter used in in vitro transcription assays. However Sso0669 appeared to inhibit transcription, although the inhibition was not sequence specific. A knockout strain of S. solfataricus PBL2025 missing the sso2273 gene was produced and used in microarray experiments in an attempt to determine the role of Sso2273 within the cell. The absence of Sso2273 appeared to have no effect on the expression of the dps-like gene, however strong repression of an operon containing genes involved in Sulphur assimilation was observed.

Viruses infecting the Archaea exhibit a tremendous amount of morphological and genetic diversity. This is especially true for crenarchaeal viruses from the family Fuselloviridae, which possess spindle-shaped capsids and genomes that harbor a great number of uncharacterized genes. The functions of these unidentified gene products are of interest as they have the potential to provide valuable insights into the fusellovirus infection cycle and archaeal viruses in general. In an effort to better characterize the genetic requirements of the Fuselloviridae, we have performed genetic and biochemical experiments using the best studied fusellovirus, Sulfolobus spindle-shaped virus 1 (SSV1). A comprehensive genetic analysis of SSV1 was conducted using long inverse PCR and transposon mutagenesis. The results of this work illustrate that SSV1 is highly tolerant of mutagenesis. A robust protocol for the purification of recombinant VP2 protein from E. coli was developed and should be useful for future studies aimed at characterizing the biochemical and structural characteristics of this SSV1 structural protein. Finally, the first insights into a fusellovirus infection are presented and provide the framework for a detailed characterization of the fusellovirus infection cycle. The results and significance of this work are presented in the chapters that follow.

Culture independent studies have revealed a greater diversity of Archaea than the two kingdoms initially defined by Carl Woese. Culturing species from the newly discovered archaeal lineages, as with the majority of microbes, has been difficult. To overcome the culturing dilemma, metagenomics is being used to reconstruct environmental genomes. Two metagenomic studies are presented in this thesis, with the aim of recovering newly proposed archaeal genomes from the Nanoarchaeota and Korarchaeota. In the first study, a sediment sample, NZ13, was collected from a terrestrial New Zealand hot spring. Along with the sediment sample, two complex enrichments were sequenced on an Illumina MiSeq platform. Assembly and differential binning recovered two nearly complete genomes of a nanoarchaeote and a korarchaeote. The NZ13 nanoarchaeote is similar to other terrestrial nanoarchaeotes, which lack an ATP synthase and encode genes for glycolysis/gluconeogenesis and archaella. One notable difference is the NZ13 nanoarchaeote contains CRISPR genes, which are absent in other terrestrial nanoarchaeotes, although present in a marine nanoarchaeote, Nanoarchaeum equitans. The NZ13 korarchaeote mirrors Candidatus Korarchaeum cryptofilum, lacking genes for de novo synthesis of purines and several cofactors, while containing an abundance of peptide transporters and amino acid fermentation pathways. The second study focused on sulfide samples collected from deep-sea hydrothermal vent fields in southwestern Pacific Ocean along the Eastern Lau Spreading Center. Ten sulfide samples were sequenced on an Illumina HiSeq platform. Small subunit ribosomal RNA genes were extracted from the metagenome reads and aligned against the SILVA Ref NR 99 123 database. The preliminary results identified which samples could be prioritized for genome reconstruction of uncultured bacterial and archaeal lineages. Three uncultured bacteria, candidate division SR1, Gracilibacteria (GN02), and Parcubacteria (OD1) were identified in several samples. Many uncultured deep-sea hydrothermal archaeal lineages were identified in all samples. In particular, korarchaeotal sequences were in high relative archaeal abundances in two samples, ABE 1 and Vai Lili-2, while few nanoarchaeotal reads were classified.

Transcription, the first step in gene expression, is a highly regulated process which relies on a multi-protein complex to occur. Among these proteins are transcription factors, including initiation and elongation factors, which play differing roles in early and late stages of transcription. The mechanisms of transition from transcription initiation to elongation are not well understood in archaea, nor are the structures of the transcription factors involved. For transcription to occur in vitro, transcription factors TATA binding protein (TBP) and Transcription Factor B (TFB) are sufficient to allow RNA polymerase (RNAP) to synthesize RNA from template DNA. Another factor, Transcription Factor E (TFE), can also join the initiation complex and is likely to be essential in vivo. TFE is known to contribute to initiation by enhancing promoter opening, and while it has been shown to persist in elongation complexes, its role after initiation is unknown. Spt4/5, the archaeal homolog of the only universally conserved RNAP-associated factor, is known to join complexes in elongation steps and enhance processivity of the polymerase. However, if Spt4/5 joins pre-initiated complexes, it has been shown to inhibit transcription activity. The experiments in this thesis show that TFE and Spt4/5 participate in a crucial interchange at the upstream fork of the transcription bubble that helps define the timing of Spt4/5 binding. Using unnatural amino acid crosslinking techniques, the points of proximity between specific regions of these two factors and the template DNA have been mapped to identify possible sites of interaction. Competitive crosslinking assays indicate the exact timing of the shift in affinity between TFE and Spt4/5 for their shared binding site on RNAP. These data, combined with transcription assays, suggest a new role for TFE in preventing premature Spt4/5 binding, corresponding with a unique localized mobility within the winged helix of TFE.