Open Access System for Information Sharing

Login Library

 

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

Interactions between Signal-Transducing Proteins Measured by Atomic Force Microscopy

Title
Interactions between Signal-Transducing Proteins Measured by Atomic Force Microscopy
Authors
김일홍
Date Issued
2011
Publisher
포항공과대학교
Abstract
Experimental analysis of biological systems by conventional methods is informative and useful, but partly limited by taking averages of molecular ensembles. This is particularly problematic when information about the behavior of an individual molecule is required. The limitation imposed on the analysis can be overcome by the measurement at a single molecular level. Therefore, new analytical techniques should be employed to gain the information on the biological systems at single molecule level. Recently, various techniques such as optical tweezers, magnetic tweezers, and atomic force microscopy (AFM) have emerged which are capable of directly measuring the biomolecular interactions at single molecular level. Among the techniques, the AFM is a well-established technique capable of high resolution imaging of individual biomolecules and measuring biomolecular interaction at the single molecule level. Because AFM is able to measure interaction force as low as the piconewton range under physiological conditions, it has been employed to study specific biomolecular interactions at the single molecule level, including DNA?DNA, antigen?antibody, and ligand?receptor interactions. Also, the sub-nanometer lateral resolution characteristic of AFM has being widely exploited for mapping the spatial distribution or localizing recognition sites of biomolecules on various surfaces. The AFM has been used to study the specific interactions between the signal-transducing proteins regulating the cellular signaling, including Munc-18-1, phospholipase D (PLD) 1, phospholipase C (PLC)-?1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Ras homologue enriched in brain (Rheb). In order to enhance the recognition efficiency and avoid undesirable multiple interactions between the protein pairs, both AFM probes and substrates were first modified with dendrons, reduced glutathione (GSH) was conjugated at the apex of each immobilized dendron, and the glutathione S-transferase (GST)-fused proteins were employed. Under the employed conditions, the probability of observing an unbinding event of the PX?(Munc-18-1), PX?SH3, and GAPDH?Rheb pairs increased, and most force?distance curves showed the single rupture events. Also, the use of the relevant dendrons (9-acid and 27-acid) of two different sizes demonstrated that the employment of the 27-acid, or in other words, the use of a larger spacing, for large biomolecules is more effective in realizing single rupture events. Dynamic force spectroscopy allowed estimation of the zero-force dissociation rate constants of the protein pairs at the single molecule level, and revealed the presence of a single potential barrier in the dissociation energy landscape. Further, the influence of a competing protein (SH3 and Munc-18-1) or metabolite (Gly-3-P) on the specific interactions was explored, demonstrating that the interactions are modulated by them. Although there are alternative conventional methodologies for obtaining such data, in particular, the ability to observe the rate of PLD1 reorganization upon the binding of a competing protein is unique. Adhesion force maps were generated to visualize individual GAPDH molecules on the surface, and showed that the number density of GAPDH decreased with the increase of Gly-3-P concentration. Maps obtained at various Gly-3-P concentrations provided information on the binding behavior. This digitized titration is expected to be applicable to any comparable system and compatible with low molecular weight metabolites. The studies show that AFM can be widely employed to elucidate protein-protein interactions and suggests that a new approach utilizing AFM for screening drug candidates would be feasible under a proper configuration.
URI
http://postech.dcollection.net/jsp/common/DcLoOrgPer.jsp?sItemId=000000895723
http://oasis.postech.ac.kr/handle/2014.oak/967
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