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Synthesis and characterization of multiple carbohydrates bearing amphiphiles: Implication of detergent flexibility for membrane protein study

Title
Synthesis and characterization of multiple carbohydrates bearing amphiphiles: Implication of detergent flexibility for membrane protein study
Author
AimanSadaf
Advisor(s)
Pil Seok Chae
Issue Date
2018-02
Publisher
한양대학교
Degree
Doctor
Abstract
Abstract Integral membrane proteins are vital for cell homeostasis of all organisms. Representing 30% of all the proteins and 60% of the total drug targets, the structure of these bio-macromolecules not only holds the key for the structure based drug design but also for the comprehension of various biochemical processes. However, the number of mapped-out integral membrane proteins is not at all parallel to their ubiquitous significance. Paucity in membrane protein structural study is a direct outcome of the incompatibility between their amphipathic nature and the requisites for bio-physical characterization. Detergents with chemical nature analogous to the lipids of membrane bilayer facilitate membrane protein study by rendering these biomolecules water soluble in a stable state. But most conventional detergents are potentially limited in stabilizing various membrane proteins outside native membranes, bringing significant attention to the development of novel detergents for membrane protein research. Detergent solubilization and stabilization efficiency is indispensable for membrane protein study. It has been empirically established that the efficacy of any detergent is a function of its architecture with major emphasis on its lipophilic and hydrophilic domains mainly. The aim of this dissertation is to rationalize the role of a linker as an important detergent tool while identifying detergent effectiveness for the selected membrane proteins by keeping DDM, the most popular classical detergent, as the standard detergent. We have developed and synthesized three classes of novel amphiphiles each divided into sub-classes based on the nature and flexibility of the linker. TRIS-derived triglucosides (TDTs) and neopentyl glycol-derived triglucosides (NDTs) are the two sub-classes of dialkylated triglucosides designed with emphasis on the difference in the rigidity of the linker functional group (amide vs ether). These two subclasses (TDTs and NDTs) not only displayed a difference in their physical properties in solution but also showed a profound different effectiveness with the tested proteins. All the amphiphiles in each sub-class were found to be superior to DDM. All TDTs outperformed than DDM while NDTs were found to be the most efficient. Among all the tested agents, NDT-C11 was found to be the best performer and was comparable to the best agent of the neopentyl glycol class, MNG-3. Enhanced efficacy conferred by the ether linkage bearing NDTs over TDTs provoked us to design amphiphiles with multiple ether linkages. To test the advantage of multiple propylene glycol units we developed, synthesized and investigated dendronic trimaltosides (DTMs). The DTMs are distinguished by the presence of a tetra-alkylated second generation hydrophobic group. The DTMs fall into two sub-sets, alkyl based dendronic trimaltosides (DTM-As) and ether based dendronic trimaltosides (DTM-Es) with a subtle difference in the linker unit at the second branch point. The lower analogue of both alkyl-based dendronic trimaltosides (DTM-A5/6) and ether-based dendronic trimaltosides (DTM-E5/6) displayed similar properties in solution. In evaluation with four model proteins, the DTM-A/Es were similarly efficient for two model proteins, but DTM-As displayed a clear superior performance for MelBSt and human β2 adrenergic receptor (β2AR). One DTM (i.e. DTM-A6) was markedly advantageous over DDM in stabilizing β2AR and its complex with Gs protein leading to a clear visualization of β2AR-Gs complex via EM. Syllitol-derived amphiphiles are designed to fabricate lipophilic and hydrophilic groups vicinal to each other over an alicyclic six-membered ring. Detergent efficacy in correlation with the effect of ethylene glycol or propylene glycerol linker for membrane protein study is the underlying objective of their design. These amphiphiles are in the process of investigation with discrete membrane proteins. Hence, in the present dissertation a systematic strategy has been employed to decipher the amphiphile proficiency for membrane protein study concerning the role of a linker in correlation with detergent architecture and solution properties. The results obtained by thorough preliminary investigation of detergent efficacy towards membrane protein solubilization and stabilization, elucidated as a function of linker, are found to be critical for rational detergent design.
URI
http://www.dcollection.net/handler/hanyang/000000105629http://repository.hanyang.ac.kr/handle/20.500.11754/68110
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIONANOTECHNOLOGY(바이오나노학과) > Theses (Ph.D.)
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