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Structure and Dynamics of the S100A5-RAGE complex and anti-CRISPR AcrIIA4 probed by NMR spectroscopy
핵자기공명 분광학을 이용한 S100-RAGE 복합체와 anti-CRISPR AcrIIA4의 구조 및 동력학 연구

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Authors
김익태
Advisor
서정용
Major
농업생명과학대학 농생명공학부
Issue Date
2018-02
Publisher
서울대학교 대학원
Keywords
AcrIIA4anti-CRISPRCas9NMR spectroscopyRAGES100A5Structure and dynamics
Description
학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2018. 2. 서정용.
Abstract
Distinct protein-protein interactions were investigated using NMR spectroscopy as well biophysical characterization tools such as calorimetry and size exclusion chromatography. In Chapter I, calcium binding of S100A5 and the interaction between S100A5 and receptor for advanced glycation end product (RAGE) were investigated using calorimetry and NMR spectroscopy. S100A5 exhibits a sequential manner to bind calcium ions using the C-terminal and N-terminal calcium binding EF-hands with the equilibrium dissociation constants (KD) of 1.3 µM and 3.5 µM, respectively. S100A5 interacts with the V domain of RAGE (RAGEv) to form a heterotrimeric complex with KD of 5.9 µM in a calcium-dependent manner, which is a distinct stoichiometry from the S100 family proteins. Chemical shift perturbation data from NMR titration experiments indicates that S100A5 employs the periphery of the dimer interface to interact with RAGEv. Distinct binding mode and stoichiometry of against different S100 family proteins could be important to modulate diverse RAGE signaling.
In Chapter II, solution structure and dynamics of AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9) widely used for genome editing were investigated. The structure of AcrIIA4 is a compact monomeric αβββαα fold consisting of three α helices flanked against three antiparallel β strands and short 310 helix. The backbone dynamics of AcrIIA4 reveals that distinct backbone dynamics in fast and slow timescales at loop regions is related to interaction surface for SpyCas9. Furthermore, AcrIIA4 binds to apo-SpyCas9 with KD ~4.8 µM and ~0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9, which infers that a binary complex between AcrIIA4 and SpyCas9 can associate with sgRNA to form a tight ternary complex, avoiding competition with the DNA substrate for SpyCas9 binding. This studies provide insights into anti-CRISPR-mediated inhibition mechanisms for disabling the SpyCas9, thereby broadening CRISPR-Cas regulatory tools for genome editing.
Language
English
URI
https://hdl.handle.net/10371/140793
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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