Confocal single molecule FRET studies of surface immobilized biomolecules

Albarghash, Alyazan; Fitter, Jörg Ludwig (Thesis advisor); von Plessen, Gero (Thesis advisor)

Aachen (2018, 2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018


Single-molecule FRET is a powerful, versatile method with widely opened frontiers going further beyond structure determination of biological macro molecules. Indeed, it facilitates monitoring molecular conformational dynamics, providing valuable knowledge that enhances our understanding of folding phenomena and other biological processes such as ligand binding kinetics. FRET can be performed in-solution on freely diffusing molecules with observation times limited to the short dwell time of the molecules thermally driven through the detection volume, typically in the millisecond regime. Alternatively, anchoring of molecules of interest to a functionally modified surface is a well-established technique to facilitate extended observation time of FRET measurements. In this work, an improved protocol of surface immobilization based on biotin-neutravidin binding assay is presented. This protocol is further employed to achieve FRET measurements on biological molecules at the single molecule level under various conditions. Once the molecules are successfully tethered on-surface, FRET observation of labeled biomolecules is then limited by the photostability of the elected fluorophores. Therefore, the photophysical properties of the Alexa488 and Alexa647 fluorophores, elected to be the FRET-pair in this work, are studied. The effect of various classes of the so-called photoprotection additives on the photophysical behavior of the elected FRET-pair is investigated in terms of emission longevity and signal stability. A protocol for the enhancement of the FRET-pair photostability in aqueous buffers, as well as, under application of denaturants is introduced. Two variants of structurally rigid double-stranded DNA oligonucleotides, with defined inter-dye distances are employed as a model system to investigate the effect of the adjusted experimental conditions on the output of the FRET measurement. On the other hand, Phosphoglycerate Kinase (PGK) is studied in a native condition and under application of guanidine hydrochloride (GdnHCl) denaturant at 0.7 M concentration. The FRET measurements are achieved with a confocal microscopy setup that facilitates resolving fluorescence lifetimes. The advantageous temporal resolution of the employed confocal setup enabled utilizing the fluorescence lifetime analysis for accurate determination of FRET efficiencies. The results of the fluorescence lifetime analysis crucially contributed to a thorough understanding of the FRET processes, as well as the associated photophysical phenomena. Consequently, the necessary calibration parameters which correct for the different detection efficiencies in different detection channels are accurately determined for each experimental condition. The observation and the analysis of one-level FRET from each of the DNA variants, exhibiting either high or low FRET, in addition to dynamical FRET from PGK are presented in this work. Additionally, FRET measurements facilitating the observation of PGK unfolding under the application of 0.7 M GdnHCl are also presented. Finally, the fluorescence lifetime analysis of donor's emission after acceptor's photobleaching revealed the existence of two possible dark-states of the acceptor, of which one exhibits a quenching effect on the donor.


  • Department of Physics [130000]
  • Chair of Experimental Physics I A and I. Institute of Physics [131110]