碩士 / 國立清華大學 / 生物技術研究所 / 91 / Plant non-specific lipid transfer proteins (ns-LTPs) are small, basic proteins which are characterized by eight strictly conserved cysteines forming four disulfide bridges. Ns-LTPs have in vitro lipid transfer and binding ability and seem to play a role in plant defense. Two main groups of ns-LTPs, ns-LTP1 and ns-LTP2, have been identified with molecular weight of about 9 and 7kDa, respectively. Ns-LTP1 and ns-LTP2 share similar structures composed of four α-helices and flexible C-terminal tail. An interesting difference between ns-LTP1 and ns-LTP2 is the mismatched disulfide linkage in CXC motif. In ns-LTP1, “X” possesses hydrophilic residue projecting outside whereas hydrophobic residue burring inside in ns-LTP2. The “X” may act as a key residue determining the disulfide bond pattern.
In this study, an expression system in E. coli was established for the production of ns-LTP2 wild-type and mutants. Recombinant ns-LTP2 is confirmed to be structural and functional identical with purified ns-LTP2. Two mutants were created for the study of structure/function analysis. Phe36 replacement of Asn was introduced for the proof of the hypothesis that the central residue in CXC motif may govern the cysteine pairing and influence the overall fold of the protein. Whether the disulfide bond pattern changed still needs determination. However, the replacement changed the conformation of ns-LTP2. Another mutant, C11A/C25A was introduced for the verifying of the stability caused by disulfide bond. The breakage of this linkage reduced the α-helix content. Both of the mutants displayed a decrease of lipid binding ability which may due to the conformation change brought by mutation.
| Identifer | oai:union.ndltd.org:TW/091NTHU0108006 |
| Date | January 2003 |
| Creators | 黃琳懿 |
| Contributors | Ping-Chiang Lyu, 呂平江 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 72 |
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