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Studying biomolecular interactions using atomic force microscopy


Studying biomolecular interactions using atomic force microscopy

Madwar, Carolin (2009) Studying biomolecular interactions using atomic force microscopy. Masters thesis, Concordia University.

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MR67235.pdf - Accepted Version


Many biological systems involve mechanical interactions and specific molecular recognition events that are essential for their function. Single molecule force spectroscopy (SMFS) is a powerful and versatile atomic force microscopy (AFM)-based technique, which allows probing such interactions at the single molecule level, with piconewton sensitivity, thereby illuminating their dynamics as well as their structural and mechanical properties. A fundamental requirement in these studies is the immobilization of biomolecules between the AFM probe and the sample surface, preferably by a long flexible molecular spacer, such as poly(ethylene glycol) (PEG). The goal of this project is to investigate the binding interactions in four distinct biomolecular systems at the single molecule level, using SMFS. A new amination strategy was used to attach a novel bifunctional PEG spacer containing amine- and thiol-reactive termini. Biomolecules under investigation were tethered to AFM tips by formation of covalent linkages to this versatile spacer. Various tests, including surface plasmon resonance and a UV-based enzyme assay, were carried out to evaluate and confirm AFM tip functionalization. SMFS of biotin/streptavidin yielded an average unbinding force of 59 pN at 4000 pN/s loading rate. SMFS of concanavalin A/mannose yielded an average unbinding force of 50 pN at loading rate of 6000 pN/s. SMFS of a de novo heterodimeric E/K coiled/coil yielded an average unbinding force of 41 pN at 7000 pN/s loading rate with a k off rate calculated to be 15.8 s-1 . SMFS of these biomolecular systems were not only successful and informative, but also provide exciting directions towards future applications.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Madwar, Carolin
Pagination:xxi,143 leaves : ill. (chiefly col.) ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Thesis Supervisor(s):Cuccia, Louis
ID Code:976737
Deposited By: Concordia University Library
Deposited On:22 Jan 2013 16:32
Last Modified:18 Jan 2018 17:43
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