Open Access System for Information Sharing

Login Library

 

Thesis
Cited 0 time in webofscience Cited 0 time in scopus
Metadata Downloads

Analysis and Application of diffusional alteration of membrane proteins using single-protein imaging

Title
Analysis and Application of diffusional alteration of membrane proteins using single-protein imaging
Authors
김동균
Date Issued
2020
Publisher
포항공과대학교
Abstract
Membrane proteins play crucial roles in communication across the cell membrane through dynamic bio-molecular interactions. More than half of proteins are involved in cellular interactions on the plasma membrane, and these interactions determine the cell fate in response to various environmental factors. Membrane proteins have a various structure in plasma membrane including single spanning transmembrane domain, seven transmembrane domains, embedding with beta barrel and peripheral proteins. And these membrane protein regulate and mediate the function including various enzymatic activity, transporting the molecules, recognition, and signal transduction. However, among these various property, diffusion of the membrane protein on the plasma membrane is one kind of physical characters. The definition of diffusion is that the net movement of molecules from a region of high concentration to a region of low concentration as a result of random motion of the molecules. Thus, using the diffusivity of target membrane protein, we could distinguish and analyze binding state and molecular reaction progresses. To elucidate the diffusivity of target membrane protein on living plasma membrane, we utilize super-resolution based single-particle tracking microscope techniques. The 405, 488, 561, and 642 nm laser line and TIRF type objective lens equipped super-resolution microscopy enables detection of multiple individual particles using photoactivatable fluorophores linked membrane protein. And we employ the EGFR family and Syntaxin1A as a model membrane protein. At the beginning, we show a diffusional mobility alteration for analyzing the interaction between membrane protein in aqueous part and ligands in the crowded membrane of living cells. That mobility shift was sensitive to the size of the binder. And also have a molecular specificity. And alteration was caused from direct binding of proteins, not the result of signal transduction. In addition, we combine diffusion coefficient distribution of each states from imaging and Reaction Progress Kinetic Analysis (RPKA). A single particle tracking-based reaction progress kinetic analysis was developed to simultaneously determine the kinetics of multiple states of protein complexes in the membrane of single living cell. The subpopulation ratios of different states were quantitatively extracted from the diffusion coefficient distribution. Using this method, we investigate the series of molecular mechanisms of EGFR induced by cetuximab. The environment of live cell membrane was extremely complex to determine the factor that related with diffusivity, so we employ the in vitro membrane and membrane protein system. Using supported lipid bilayer (SLB) system, we prove the effect of the concentration and size of extracellular domain of membrane protein in diffusion. In diluted membrane protein situation, the result of experiment and simulation shows similar as a Saffman and Delbrück model. However, in crowded and complex situation, the diffusion of membrane protein decreased after molecular binding similar to previous live cell experiment. Lastly, I apply the imaging technique to drug screening field. Screening drug candidates rapidly is the first step for developing new pharmaceutical drugs. One of the promising ways to reduce the screening steps and cost is to use directly living cells for screening, instead of using purified target proteins. The compounds screened using living cells will have more biologically activities than those screened from in vitro assays. Here I report a robust method for screening drug candidates in a living cell based on single-protein imaging. I tested three different membrane proteins of epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 and found effective natural compounds for each protein. The screening method I introduce will be widely used for screening the potential drug candidates using a living cell.
URI
http://postech.dcollection.net/common/orgView/200000372494
https://oasis.postech.ac.kr/handle/2014.oak/111091
Article Type
Thesis
Files in This Item:
There are no files associated with this item.

qr_code

  • mendeley

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Views & Downloads

Browse