Seminar “Seeing biological macromolecules in action: From ensemble to single molecule”
Date
Thursday, 23 June 2016
Time
2:00 pm
Place
University of Barcelona
Faculty of Physics Building
Room 3.20, 3rd floor
Speaker
Dr Namik Akkilic, Mesa+ Institute for Nanotechnology, University of Twente (The Netherlands)
Abstract
The study of electron transfer reactions in redox proteins are not only crucial for many physiological functions but also contribute to applications in biomolecular electronics and biosensing. I will describe an optical method for the detection of the redox state of a metalloprotein, azurin, based on excited state energy transfer (FRET) from a covalently attached Cy5 molecule to the protein redox center (copper).
The fluorescence intensity of a single azurin molecule shows an “on” and “off” switching as a function of time which can be controlled externally using 2 sensitive methods: (i) Chemically-induced (1) and (ii) Electrochemically-induced switch (2, 3). To conclude, we were able to make a quantitative assessment of the heterogeneity of the reaction kinetics and redox thermodynamics for individual azurin molecules with both covalently immobilized on a passive surface (i) and on gold electrode (ii).
In the second part, I will talk about smart polymer/protein architectures that are covalently attached by one end to a substrate. Total internal reflection fluorescence (TIRF) and confocal microscopy allowed us to study the fluorescence switching behaviour of biomolecules down to the single molecule level and we demonstrate that this switching is both fast and random. We proposed this system as a platform for switchable sensor applications, but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush. Moreover, we support our experimental results with numerical self-consistent field (nSCF) theory which enabled us to predict the location of a single protein molecule that highly depends on the size of the molecule, polymer chain length, and the brush density (4).
1. Akkilic N, Van Der Grient F, Kamran M, Sanghamitra NJM. “Chemically-induced redox switching of a metalloprotein reveals thermodynamic and kinetic heterogeneity, one molecule at a time”. Chemical Communications. 2014; 50(93):14523-6.
2. Akkilic N, Kamran M, Stan R, Sanghamitra NJM. “Voltage-controlled fluorescence switching of a single redox protein”. Biosensors and Bioelectronics. 2014.
3. Salverda JM, Patil AV, Mizzon G, Kuznetsova S, Zauner G, Akkilic N, et al. “Fluorescent cyclic voltammetry of immobilized azurin: Direct observation of thermodynamic and kinetic heterogeneity”. Angewandte Chemie – International Edition. 2010; 49(33):5776-9.
4. Akkilic N, Leermakers FAM, de Vos W. 2015, “Responsive polymer brushes for controlled nanoparticle exposure”. Nanoscale, 7, 17871-78
