Conformational distribution of protein molecule revealed by single-object cryo-EM sampling = 저온 전자현미경 이미지 샘플링을 통한 단일 단백질 분자의 통계적 구조 분포 연구

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Investigation of the structure of the protein molecule is essential for understanding the function of the protein. A numerous number of studies have been conducted so far to reveal three-dimensional structures of various proteins. However, studies on the conformational distribution of the protein molecule that can possess have not been extensively issued due to the lack of experimental methods. Recently, a class of proteins that function in unstructured states, called Intrinsically Disordered Protein (IDP), as well as proteins with compact structures play important roles in various biological processes. The qunatitative conformational distribution of unstructured protein molecules is meaningful information for understanding biological processes involving these proteins. However, the inherent flexible structures of these proteins make it difficult to experimentally determine the conformational distribution. Here, we present a method named “Single-Object Cryo-EM Sampling (SOCS)” to study the conformational distribution by applying site specific nanogold labeling. Two nanogold particles were labeled at two desired target sites on cytochrome c and the distance between two particles was measured as a structural parameter via cryo-electron microscopy. The key is that the concept of single-object scattering sampling (SOSS) idea is applied here so that the distance information can be extracted from each single protein molecule. The contrast provided by the nanogold particles is strong enough to provide the distance between them although the protein itself is not visible due to the low contrast. A number of cryo-electron microscopy images of nanogold labeled cytochrome c for both folded and unfolded states were obtained to extract a statistical distribution of interparticle distance, which is associated with the shape of conformational distribution. While conventional cryo-electron microscopy provides opportunities to reconstruct the averaged three-dimensional structure of protein complexes with molecular weights higher than 100 kDa, the experimental concept used in this study can detect various conformations of the protein including rarely-populated structures even for small or disordered proteins. Data processing procedures and analysis scheme on statistical distribution were also developed and discussed. This experimental concept is expected to have a potential applicability to study the conformational distribution of objects with flexibly varying structures and their participating reactions at a single molecule level.
Advisors
Ihee, Hyotcherlresearcher이효철researcher
Description
한국과학기술원 :화학과,
Publisher
한국과학기술원
Issue Date
2018
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 화학과, 2018.8,[viii, 89 p. :]

Keywords

protein conformation▼asingle molecule▼acytochrome c▼ananogold labeling▼acryo-electron microscopy▼asingle-object sampling; 단백질 구조▼a단일 분자▼a사이토크롬-씨▼a금 나노 입자 표지▼a저온 전자현미경법▼a단일 대상 샘플링

URI
http://hdl.handle.net/10203/265492
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=828284&flag=dissertation
Appears in Collection
CH-Theses_Ph.D.(박사논문)
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