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Structural Studies on SAV0324 from Staphylococcus aureus by X-ray Crystallography : X-선 결정학에 의한 황색포도상구균 유래 SAV0324 단백질의 구조 연구
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 이봉진 | - |
dc.contributor.author | 구지성 | - |
dc.date.accessioned | 2017-10-31T08:19:43Z | - |
dc.date.available | 2020-10-06T08:34:11Z | - |
dc.date.issued | 2017-08 | - |
dc.identifier.other | 000000146063 | - |
dc.identifier.uri | https://hdl.handle.net/10371/137934 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 약학대학 약학과, 2017. 8. 이봉진. | - |
dc.description.abstract | Staphylococcus aureus is a gram positive bacteria which
produces enzymes, toxins, and small RNAs that are often harmful to the body. About 20-30% of the human population are carriers of the bacteria and the bacteria is responsible for causing many diseases and casualties worldwide. SAV0324 is a glycine cleavage system H-like protein (GcvH-L) which acts as a carrier for lipoyl moiety. SAV0324 undergoes lipoylation by SAV0327 protein and subsequently undergoes ADP-ribosylation process. Without ADP-ribosylation, the virulence of Staphylococcus aureus bacteria decreases and therefore, inhibition of lipoylation would be crucial. Using Structure Based Drug Design (SBDD) as a background knowledge, the structure of SAV0324 protein was determined at 1.88Å. Specifically, SAV0324 conserved five alpha-helices and nine beta-strands in its structure and the site of lipoylation in SAV0324 was already determined to be E53 (53rd glutamate) and K56 (56th lysine) residues by other researchers. Furthermore, crystallization of SAV0327 protein was successful. Through binding test between SAV0324 and SAV0327, the results showed the two proteins did not have direct binding interaction in the absence of lipoic acid. Thus, in addition to SAV0324s structural information, determination of SAV0327 protein structure and its active site would be of great significance. Knowing both SAV0324 and SAV0327 thoroughly would be critical in controlling and understanding bacterial virulence of Staphylococcus aureus. | - |
dc.description.tableofcontents | I. General Introduction ············································ 1
1.1 Staphylococcus aureus ··········································· 1 1.2 SAV0324 ········································································· 2 1.3 Structure Based Drug Design ···························· 3 II. Purpose of Study ················································· 5 III. Materials and Methods ···································· 6 2.1 Materials ········································································ 6 2.1.1 Reagents ···········································································6 2.1.2 Apparatus ·········································································6 2.2 Methods ·········································································· 7 2.2.1 Cloning ··············································································7 2.2.2 Overexpression Test ·····················································8 2.2.3 Solubility Test ································································9 2.2.4 Affinity Chromatography (IMAC) ·····························9 2.2.5 Size Exclusion Chromatography (SEC) ··················· 10 2.2.6 Dialysis and Concentration ··········································11 2.2.7 MALDI-TOF ···································································12 2.2.8 Circular Dichroism (CD) ··············································12 2.2.9 Crystallization ··································································13 2.2.10 Structure Determination ···············································13 IV. Results ·································································· 14 3.1 Polymerase Chain Reaction (PCR) ··················· 14 3.2 Overexpression and Solubility ···························· 15 3.3 Purification (IMAC) ················································ 17 3.4 MALDI-TOF ······························································ 18 3.5 Circular Dichroism (CD) ······································· 19 3.6 Purification (SEC) ···················································· 20 3.7 Crystallization ···························································· 21 3.8 Structure Determination ········································ 22 3.8.1 SAV0324 Structure ··························································22 3.8.2 Crystal Data Collection & Refinement ·······················24 3.8.3 Ramachandran Plot ··························································25 3.8.4 Topology ·············································································26 3.8.5 Homology Search ·····························································27 3.9 Active Site ·································································· 29 3.10 Binding Test ······························································ 30 3.11 SAV0327 ······································································· 31 3.11.1 Cloning, Overexpression, and Solubility ·················· 31 3.11.2 Purification (IMAC & SEC) ········································34 3.11.3 Crystallization ··································································36 V. Discussion ····························································· 38 VI. References ··························································· 41 국문초록 ········································································ 44 | - |
dc.format | application/pdf | - |
dc.format.extent | 1041025 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Structure Based Drug Design (SBDD) | - |
dc.subject | Staphylococcus aureus | - |
dc.subject | X-ray crystallography | - |
dc.subject | SAV0324 | - |
dc.subject | lipoylation | - |
dc.subject | virulence | - |
dc.subject.ddc | 615 | - |
dc.title | Structural Studies on SAV0324 from Staphylococcus aureus by X-ray Crystallography | - |
dc.title.alternative | X-선 결정학에 의한 황색포도상구균 유래 SAV0324 단백질의 구조 연구 | - |
dc.type | Thesis | - |
dc.description.degree | Master | - |
dc.contributor.affiliation | 약학대학 약학과 | - |
dc.date.awarded | 2017-08 | - |
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