Collagen is an important structural protein in living organisms and plays an indispensable role in connecting cells and tissues, such as in musculature, bone, and ligament. The stability and conformation of collagen are, however, strongly influenced by ambient temperature and constitutes an interesting subject of study. Thermally induced conformation change of collagen has been investigated by techniques such as differential scanning calorimetry (DSC) and second harmonic generation. DSC is a powerful method in uncovered important thermal dynamics properties including phase change, enthalpy, and thermal stability of the collagen. However, due to its collective nature, no localized information can be found. For comparison, second harmonic generation, which reflects structural symmetry, can be combined with laser scanning microscopy to investigate localized variation. It has been shown in previous studies that the thermal stability of collagen is strongly influenced by the water content within collagen. For comparison, we are investigating the conformational change of collagen under a vacuum stat with second harmonic microscopy so as to isolate environmental effects, particularly those from water and oxygen. In this way, we have found the conformational change of collagen takes place at a much higher temperature and activation energy. Additionally, the high spatial resolution achieved also allows many further possibilities.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21819号 / 農博第2332号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5191(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

PagP is an integral outer membrane enzyme that transfers a palmitoyl group from a phospholipid to lipid A and the polar headgroup of phosphatidylglycerol (PG). Palmitoyl-lipid A and palmitoyl-PG (PPG) have been implicated in resistance to host immune defenses. PagP proteins are diverse, the E. coli PagP belongs to the major clade of PagP homologs and palmitoylates lipid A regiospecifically at the 2-position, whereas P. aeruginosa PagP belongs to the minor clade of PagP homologs and instead palmitoylates lipid A regiospecifically at the 3’-position. Our objective was to understand how PagP has been adapted in nature to interact with multiple lipid substrates and products. We investigated the structure-function relationships of key major clade homologs, to show that Bordetella PagP palmitoylates lipid A at the 3’-position and employs surface residue T29 in its palmitoyltransferase reaction. Legionella PagP palmitoylates lipid A at the 2-position and was confirmed to select a palmitate chain from a pool including iso-methyl branched phospholipids characteristic of this species. PagP is usually encoded as a single copy on the chromosome in most bacteria, but two copies of pagP are found in endophytic bacteria. These duplicated PagP homologs from the major clade branch into two subclades, namely chromosomal and plasmid-based PagP homologs. The chromosomal PagP homologs exhibit interacting periplasmic D61 and H67 residues, which are naturally mutated in plasmid-based PagP homologs, and are associated with a conformational change in the -barrel that determines its ability to palmitoylate PG. Chromosomal PagPs can convert PPG to bis(monoacylglycero)phosphate (BMP) and lysophosphatidylglycerol (LPG) through a periplasmic active site controlled by the invariant Y87 residue of E. coli PagP. Plasmid-based PagP homologs appear to have been adapted instead as monofunctional lipid A palmitoyltransferases. These results points to a common ancestor for PagP proteins. Knowledge gained from these studies can be applied to protein engineering. / Thesis / Doctor of Philosophy (PhD)

"An apparatus for the delivery of radiation to the tip-sample interface of an Atomic Force Microscope (AFM) is demonstrated. The Pulsed Light Delivery System (PLDS) was fabricated to probe photoinduced conformational changes of molecules using an AFM. The PLDS is 67 mm long, 59 mm wide, and 21 mm high, leaving clearance to mount the PLDS and a microscope slide coated with a thin film of photoactive molecules beneath the cantilever tip of a stand-alone AFM. The PLDS is coupled into a fiber pigtailed Nd:Yag frequency doubled laser, operating at a wavelength of 532 nm. The radiation delivered to a sample through the PLDS can be configured for continuous or pulsed mode. The maximum continuous wave (CW) power delivered was 0.903 mW and the minimum pulse width was 12.3 ms (maximal 401 ms), corresponding to a minimal energy of 0.150 nJ (maximal 362 nJ), and had a cycle duration of 10.0 ms. The PLDS consists of micro-optical components 3.0 mm and smaller in diameter. The optical design was inspired by the three-beam pick-up method used in CD players, which could provide a method to focus the pulse of light onto the sample layer. In addition, the system can be easily modified for different operational parameters (pulse width, wavelength, and power). As proof that the prototype design works, we observed a photoinduced ‘bimetallic’ bending of the cantilever, as evidenced by observing no photoinduced bending when a reflective-coated cantilever was replaced by an uncoated cantilever. Using the apparatus will allow investigation of many different types of molecules exhibiting photoinduced isomerization."