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Development of novel saccharide-based amphiphiles as chemical tools for membrane protein analysis

Title
Development of novel saccharide-based amphiphiles as chemical tools for membrane protein analysis
Other Titles
막단백질 연구를 위한 화학적 툴로써 탄수화물 기반의 새로운 양친매성 분자의 개발
Author
Muhammad Ehsan
Advisor(s)
Pil Seok Chae
Issue Date
2018-02
Publisher
한양대학교
Degree
Doctor
Abstract
Membrane proteins are of main importance to living cells. These bio-macromolecules are involved in many cellular processes such as material transport, signal transduction and regulation of cell-cell contacts to maintain cellular homeostasis and to ensure coordinated cellular activity in all organisms. Numerous human diseases such as cancer, cystic fibrosis, epilepsy, heart failure, hypertension, and Alzheimer diseases are associated with membrane proteins dysfunction, indicating that information on membrane protein structures is critically important in drug development. Despite the biological and pharmaceutical importance of membrane proteins, their structural and functional characterizations are largely hampered mainly by their insolubility in aqueous solution and difficulty associated with obtaining sufficient amounts of protein materials in a high purity. The most widely used agents for membrane protein manipulation are detergents, amphipathic molecules bearing hydrophilic head and hydrophobic tail groups. However, conventional detergents have limitation in stabilizing membrane proteins, particularly eukaryotic proteins and membrane protein complexes. Therefore, it is necessary to develop novel amphipathic agents with enhanced membrane protein stabilization efficacy to facilitate membrane protein structural and functional study. In this thesis, we will introduce several novel classes of amphiphiles developed for membrane protein structural study. These amphiphiles bear a new hydrophilic group and/or a new hydrophobic group, thereby having architecture distinct from conventional detergents. To assess the utility as a biochemical tool, these agents were evaluated for solubilization and stabilization of a diverse set of membrane proteins. The first class of novel detergents we developed is penta-saccharide-bearing amphiphiles. These agents contain a highly branched penta-saccharide head group connected to a branched hydrophobic group directly (PSAs) or with an ether linkage (PSEs). The PSA/Es displayed enhanced membrane protein stability compared to conventional detergents, indicating a positive role of the highly branched penta-saccharide head group in retaining the native conformations of membrane proteins. In addition, protein-detergent complexes (PDCs) formed by PSA/Es were smaller and more homogeneous than those formed by n-dodecyl-β-D-maltoside (DDM), indicating that the new agents would be useful in membrane protein structural studies. The second class of novel detergents we designed is tripod penta-saccharide amphiphiles (TPSs). These agents are different from PSA/Es by an increase in the number of the alkyl chains from two to three and a structural modification of the penta-saccharide head group by inserting a propylene spacer between the central and peripheral glucose units. These structural variations allowed us to prepare three sets of the TPSs with distinctive structures. Some of the TPSs were effective at stabilizing membrane proteins (complexes) tested here compared to a conventional detergent and use of a representative TPS led to clear visualization of a G protein-coupled receptor (GPCR)-Gs complex via electron microscopy (EM) analysis, indicative of profound potential in membrane protein research. The next class of detergents introduced is steroidal-based penta-saccharide amphiphiles (SPSs). These amphiphiles share the previously developed penta-saccharide head group with each other, but variation in the hydrophobic group was attained by utilizing four different steroidal groups (cholestanol, cholesterol, sitosterol and diosgenin), either with or without use of a propylene linker between the head and tail groups. When we evaluated detergent ability to stabilize membrane proteins, some SPSs showed the marked ability to stabilize a couple of membrane proteins compared to conventional detergents. Finally, vitamin E-bearing glycosides (VEGs) were developed as a novel tool for membrane protein study. A natural product (i.e., vitamin E) and three different saccharide units (i.e., branched triglucoside, branched dimaltoside and penta-saccharide) were used as the hydrophobic and hydrophilic groups, respectively. These agents were tested with a set of membrane proteins to investigate detergent capability of solubilizing and stabilizing membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. This result reveals a pivotal importance of the chain length and molecular geometry of detergent hydrophobic group in determining detergent efficacy for membrane protein stabilization.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/68098http://hanyang.dcollection.net/common/orgView/200000431987
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIONANOTECHNOLOGY(바이오나노학과) > Theses (Ph.D.)
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