Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
Monday 13 June 2016
to 16:00 at
Vlodomyr Chmyrov - KTH, Dept. of Applied Physics; Biomolecular Physics
PhD Thesis: Fluorescence spectroscopy and imaging have a very broad spectrum of applications within the life sciences, in particular for detection and characterization of biomolecular dynamics and interactions in different environments. This thesis comprises projects that strive to further expand the information content extracted from the detected fluorescence, leading to sensitive readout parameters for studies of biomolecular dynamics and interactions. Two major strategies are presented to achieve this aim. The first strategy is based on the expansion of the available readout parameters beyond the "traditional" fluorescence parameters: intensity, wavelength, polarization and fluorescence lifetime. The additional parameters are based on blinking properties of fluorescent labels. In particular on transitions between singlet and triplet states, and transitions between the trans- and cis-isomers of fluorophores. Two publications in the thesis are based on this strategy (paper I and IV). The second strategy is based on the utilization of fluorescence intensity fluctuations in order to detect the oligomerization mechanisms of fluorescently labeled peptides and proteins. This strategy combines the intensity fluctuation analysis and the readout of distance dependent energy transfer between fluorescent molecules together with the correlation analysis of fluorescence from two labeled proteins emitting at different wavelengths. Another two publications presented in the thesis are based on the second comprehensive strategy (papers II and III).
In the paper I, Fluorescence Correlation Spectroscopy (FCS) and Transient State (TRAST) imaging were applied to follow the isomerization kinetics of the Merocyanine 540 fluorophore incorporated into lipid membranes. It's isomerization kinetics is highly depend on the local viscosity, and thus could be used to characterize the membrane fluidity of lipid vesicles and live cell membranes. Paper II presents a new FCS-based approach of ultra high sensitivity to detect binding of proteins in the presence of a high fraction of proteins that do not bind to each other. The method combines FCS with Förster Resonance Energy Transfer (FRET). The study analyzed peptides that are believed to cause Alzheimer's disease. Peptides, labeled with a fluorophore, are excited by a focused laser beam and can transfer this excitation energy by FRET to peptides labeled with another type of fluorophore if the peptides are bound to each other. The FCS analysis of fluorescence fluctuations from the fluorophore accepting the energy can reveal the oligomerization of peptides with a very high sensitivity. Paper III is a comprehensive investigation of a spider silk protein oligomerization process. It proposes a novel 3-steps mechanism to explain the formation of spider silk filaments. The study is based on a range of biophysical methods. Fluorescence cross-correlation spectroscopy (FCCS) measurements added an important contribution by monitoring the covariance in fluorescence fluctuations from the two different labels, attached to spider's web proteins, providing a direct demonstration of the pH-dependent dimerization process. This dimerization underlies the entire formation mechanism of the spider threads. Paper IV}shows how blinking kinetics caused by singlet-triplet transitions in 7-nitro benz-2-oxa-1,3-diazole-4-yl (NBD) can be used to obtain information about interactions of lipids in a biological membrane. Blinking kinetics is capable to offer a more sensitive readout than conventional fluorescence-based methods, and also indicates a folding behavior of the aliphatic chains of examined lipids, which has not been detected in previous studies.
In summary, the work presented in this thesis shows that the blinking kinetics of fluorescent labels contain significant information that can be exploited by a combination of fluctuations analysis with distance dependent excitation energy transfer between the fluorescent molecules, or by analysis of fluorescence covariance between molecules that emit at different wavelengths. These fluorescence-based methods have a significant potential for molecular interaction studies in the biomedical field.