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Lee Chi-Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 154-167). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.II / Abstract --- p.IV / 摘要 --- p.VI / Content --- p.VIII / Abbreviations --- p.VIX / List of figures and tables --- p.XVII / Chapter Chapter 1 - --- Introduction --- p.1 / Chapter 1.1 --- How are the thermophilic proteins stabilized? --- p.2 / Chapter 1.1.1 --- Hydrophobic interactions --- p.2 / Chapter 1.1.2 --- Hydrogen bonds --- p.4 / Chapter 1.1.3 --- Electrostatic interactions --- p.6 / Chapter 1.1.4 --- Reduction in ΔCP --- p.9 / Chapter 1.2 --- Models of study: Thermococcus celer and yeast L30e --- p.12 / Chapter 1.2.1 --- Thermococcus celer ribosomal protein L30e --- p.12 / Chapter 1.2.2 --- Yeast ribosomal protein L30e --- p.13 / Chapter 1.2.3 --- Comparison between the two proteins --- p.13 / Chapter 1.3 --- Objective of this study --- p.20 / Chapter Chapter 2 - --- Materials and Methods --- p.21 / Chapter 2.1 --- General techniques --- p.21 / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5α and BL21(DE3)pLysS --- p.21 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.22 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.24 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.24 / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) --- p.25 / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.26 / Chapter 2.1.7 --- Ligation of DNA fragments into vector --- p.26 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.28 / Chapter 2.1.9 --- Native-PAGE electrophoresis --- p.32 / Chapter 2.2 --- Protein Engineering of Proteins --- p.35 / Chapter 2.2.1 --- Polymerase chain reaction (PCR) --- p.35 / Chapter 2.2.2 --- Site-directed mutagenesis of T. celer L30e --- p.37 / Chapter 2.2.3 --- Protein engineering of yeast L30e --- p.42 / Chapter 2.3 --- "Sub-cloning of mutation PCR fragment into expression vector, pET8c" --- p.45 / Chapter 2.4 --- Expression of recombinant proteins --- p.45 / Chapter 2.5 --- Purification of T. celer and its mutants --- p.46 / Chapter 2.5.1 --- Extraction of proteins by sonication --- p.46 / Chapter 2.5.2 --- Purification by ion-exchange chromatography --- p.47 / Chapter 2.5.3 --- Purification by affinity chromatography --- p.48 / Chapter 2.5.4 --- Purification by size exclusion chromatography --- p.49 / Chapter 2.6 --- Purification of yeast L30e and its mutants --- p.49 / Chapter 2.6.1 --- Extraction of proteins by sonication --- p.49 / Chapter 2.6.2 --- Purification yeast L30e variants by washing the inclusion bodies --- p.50 / Chapter 2.6.3 --- Purification by column chromatography --- p.51 / Chapter 2.7 --- Thermodynamic studies of proteins --- p.52 / Chapter 2.7.1 --- Guanidine-induced denaturation --- p.52 / Chapter 2.7.2 --- Thermal-induced denaturation --- p.53 / Chapter 2.7.3 --- Determination of protein stability curves by denaturant unfolding --- p.54 / Chapter 2.7.4 --- ΔCP and protein stability curve determination by thermal unfolding --- p.55 / Chapter 2.8 --- Media and buffer recipes --- p.56 / Chapter 2.8.1 --- Medium for bacterial culture --- p.56 / Chapter 2.8.2 --- Reagents for competent cell preparation --- p.58 / Chapter 2.8.3 --- Nucleic acid electrophoresis buffers --- p.58 / Chapter 2.8.4 --- Buffers for T. celer L30e variants purification --- p.59 / Chapter 2.8.5 --- Buffers for yeast L30e variants purification --- p.59 / Chapter 2.8.6 --- Reagents of SDS-PAGE --- p.60 / Chapter Chapter Three - --- Purification of T. celer and Yeast L30e --- p.63 / Chapter 3.1 --- Purification of T. celer L30e and its mutants --- p.63 / Chapter 3.2 --- Purification of yeast L30e and its mutants --- p.72 / Chapter Chapter Four - --- Thermodynamic Studies of T. celer and Yeast L30e --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Result --- p.79 / Chapter 4.3 --- Discussion --- p.85 / Chapter Chapter Five- --- Mutagenesis Study of a Charge Cluster in T. celer L30e --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Result --- p.92 / Chapter 5.3 --- Structure determination of T. celer L30e mutants --- p.99 / Chapter 5.4 --- Discussion --- p.105 / Chapter Chapter Six - --- Alanine Scanning Mutagenesis of Charge Residues of T. celer L30e --- p.114 / Chapter 6.1 --- Introduction --- p.114 / Chapter 6.2 --- Result --- p.114 / Chapter 6.3 --- Discussion --- p.121 / Chapter Chapter Seven - --- Protein Engineering of T. celer and Yeast L30e --- p.132 / Chapter 7.1 --- Introduction --- p.132 / Chapter 7.2 --- Result --- p.136 / Chapter 7.3 --- Discussion --- p.138 / Chapter Chapter Eight - --- Concluding Remarks --- p.141 / Appendix --- p.143 / Reference --- p.154

A crystal structure of OFP has been solved at 2.0 A resolution. It reveals that OFP is tetrameric and exists as a dimer of homo-dimer. It adopts the characteristic 11-stranded beta-can structure of GFP-like proteins. Based on the crystal structure, four special mutants were created primarily by site-directed random mutagenesis and error-prone PCR techniques. The first mutant is named Y37F in which a substitution of tyrosine (Y37) for phenylalanine creates a green emitter with an excitation maximum of 480 nm. The emission spectrum of Y37F is similar to that of EGFP and peaked at 498 nm. The second mutant is named K79R in which a substitution of lysine (K79) for arginine creates a red emitter with an excitation maximum of 548 nm. While the excitation maximum of K79R was literally unchanged with respected to OFP, the two emission maxima of K79R are both red-shifted to 512.5 nm and 583 nm. The third mutant is a dimeric mutant named OFP2 with W118K and V120E mutations. The absorption spectrum of OFP2 is identical to that of OFP. The emission spectrum of OFP2 has two emission maxima at 498 nm and 568 nm, respectively. Only bright orange fluorescence maximum at 568 nm is observed when OFP2 is excited at 515 nm or above. When OFP is excited between 430 nm and 470 nm, however, roughly equal amount of green emission maximum at 498 nm is also observed. The fluorescence quantum yield of OFP2 is about the same as OFP. The last mutant is named OFPm. It is a monomeric mutant of OFP2. A total of seven extra mutations (T141A/T144H/F155V/Y188P/K192S/R194S1N199A) were introduced on OFP2. The absorption maximum of OFPm is slightly red-shifted to 560 nm as compared to OFP2. The emission spectrum of OFPm has two emission maxima at 504 nm and 576.5 nm, respectively. Only red fluorescence maximum at 576.5 nm is observed when OFPm is excited at 525 nm or above. When OFPm is excited between 430 nm and 470 nm, however, the emission is mainly green fluorescence peaked at 504 nm with small amount red light peaked at 576.5 nm. The fluorescence quantum yield of OFPm is about 0.32. / A novel Orange Fluorescent Protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. This protein consists of 222 amino acid residues with the calculated molecular mass of 25.1-kDa. A BLAST protein sequence homology search revealed that native OFP has 81% sequence identity to Cerianthus membranaceus green fluorescent protein (cmFP512), 38% identity to Entacmaea quadricolor red fluorescent protein (egFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-Orange fluorescent protein (KO), and a mere 21% identity to Aequorea victoria green fluorescent protein (GFP). Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 483 and 515 nm. Bright orange fluorescence maximum at 568 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 500 nm, a considerable amount of green emission maximum at 498 nm was also observed. It has a fluorescence quantum yield (phiF) of 0.64 at 25°C. The molar absorption coefficients (epsilon) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M-1 · cm -1, respectively. Its fluorescent brightness (epsilon · phi F) at 25°C is 38,400 M-1 · cm -1. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12. OFP was readily expressed as soluble protein in Escherichia coli at 37°C. / Ip, Tsz Ming. / "November 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4733. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 139-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Cold shock proteins (Csp) are small acidic proteins that fold into ??-barrel structures with five anti-parallel ??-strands and are involved in essential cellular processes. Upon temperature downshift the synthesis of Csp proteins is drastically increased to enable cells to restore growth in the cold. These proteins facilitate transcription and translation at low temperature by functioning as RNA chaperones. Csp proteins have been most extensively studied in Bacteria but very few Csp homologues have been identified and studied in Archaea. This is the first study examining structural, functional and biophysical properties of Csp from the Antarctic archaeon Methanogenium frigidum. The fastidious growth requirements of M. frigidum make it difficult to cultivate, therefore recombinant methods have been developed for the expression and characterization of the protein. The analysis by transverse urea gradient gel electrophoresis (TUG-GE) revealed that M. frigidum Csp folds by a reversible two-state mechanism and has a low conformational stability. The spectroscopic analysis of the protein performed by Circular Dichroism (CD) spectroscopy disclosed features typical of other homologous proteins. A possible association between Csp and RNA has been proposed according to MALDI-TOF mass spectrometry analysis. The effect of a Nterminal polyhistidine affinity tag on the biophysical properties of Csp was also examined. The biological activity of Csp was investigated by complementation of an E. coli cold sensitive mutant. These studies revealed that the M. frigidum Csp is biologically active and can function in E. coli.

Thesis (Ph. D.)--Lehigh University, 2002. / Includes bibliographical references and vita.