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Title:Advancing fast relaxation imaging to determine protein stability and folding kinetics on poly(n-isopropyl acrylamide) films
Author(s):Mora Sierra, Zully A
Advisor(s):Leckband, Deborah E
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):Protein stability
polymer films
Abstract:Several studies have reported the use of the thermo-responsive polymer PNIPAM to capture and release cells or proteins for medical purposes; however, this assumes that PNIPAM does not perturb the stability of biomolecules. Previous analytical and spectroscopic techniques have not been able to confirm this assumption due to limitations in detection of proteins on surfaces. Additionally, protein absorption above the lower critical solution temperature of PNIPAM (32℃) is dependent on the grafting density and molecular weight of the end grafted PNIPAM. The work presented here applies a novel technique to demonstrate how protein adsorption on PNIPAM affects protein thermodynamic stability and kinetics. Fast Relaxation Imaging (FReI) was applied to quantify the folding stability and dynamics of proteins in contact with PNIPAM at varying grafting conditions categorized as brush, weakly overlapping, and mushroom regime. FReI detects protein unfolding in situ by imaging changes in fluorescence resonance energy transfer (FRET) after fast temperature perturbations. The protein used in experiments is phosphoglycerate kinase (ePGK1), which undergoes cooperative unfolding. Contrary to previously accepted notions, the melting temperature and kinetic parameters of ePGK1-FRET reveal that the weakly overlapping and mushroom regimes do in fact perturb the thermodynamic stability and kinetics of ePGK1. Similar to the protein adsorption trend, the perturbations exhibit a bell-shape dependence on the PNIPAM regimes. Additionally, this work shows that FReI can be used to confirm how biomaterials perturb proteins on polymer surfaces.
Issue Date:2020-12-11
Type:Thesis
URI:http://hdl.handle.net/2142/109543
Rights Information:Copyright 2020 Zully Mora Sierra
Date Available in IDEALS:2021-03-05
Date Deposited:2020-12


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