Glycine receptor a3K/L
The glycine receptor (GlyR) is a protein involved in neuron communication. Upon binding of glycine it transports chloride ions, thereby fine-tuning neuron activity. Structurally, the GlyR is a pentamer assembled from one or more different GlyR subtypes. Its diversity is further increased by alternative splicing and RNA editing. Dysfunction of the GlyR is linked to neurological disorders. Furthermore, the alpha 3 type GlyR is a promising target to treat pain, but more fundamental insights on receptor structure and function are needed.
In our research we:
- determine stoichiometry and co-assembly of differentGlyRα3 isoforms at single-receptor resolution
- apply multi-color fluctuation imaging methods to probe co-assembly at the ensemble level.
- design FRET-active GlyRs to probe conformation and study the mechanism of channel gating.
- correlate simultaneously recorded information from electrophysiology and smFRET experiments
Starting with in vitro and cell-culture based fluorescence correlation spectroscopy investigations of the human immunodeficiency virus (HIV) Integrase protein and the human HIV cofactor LEDGF/p75 more than 10 years ago, currently we are able to fluorescently label and track individual HIV and murine leukemia virus (MLV) virions during the viral replicative cycle, and at the same time assess the quaternary structure of the IN protein via Förster resonance energy transfer (for a nice review, see Parveen et al., 2018). On the one hand, we continuously develop novel single virus microscopy tools to interrogate the virions and intracellular viral complexes in more detail, and on the other hand we try to solve timely questions in retrovirology research together with KU Leuven virologist Prof. Zeger Debyser and colleagues.
Illustration of single virus (HIV/MLV) imaging. Partially adapted from Borrenberghs et al., 2016.
Dynamic structural biology
Single-molecule based Förster resonance energy transfer can be used to measure in real time the distance between two fluorescent probes that are attached to a biomolecule with near Ångstrom precision. The main advantages of this approach are that the biomolecule does not need to be crystallized, does not need to be purified in large amounts, and can be assessed in real time in solution. On the one hand, we use this method to gain mere structural insights into proteins, and on the other hand, we use it to provide detailed insights into the intrinsic structural dynamics that some proteins possess.
Illustration of biological systems investigated with smFRET. EF-Tu data adapted from Talavera et al., 2018. CcdA data adapted from Burger et al., 2017.
Multiparameter Fluctuation imaging
Advanced image correlation spectroscopy allows extracting maximal information from live samples containing diffusing molecules. We combined pulsed interleaved excitation with raster image correlation spectroscopy, which allows exploiting the fluorescence lifetime to discern between different fluorescent species or to remove noise from experimental data. We developed a new image correlation algorithm that allows studying molecular mobility in arbitrarily shaped regions of interest (e.g. the membrane or other organelles). We also developed an algorithm to resolve different diffusing molecules on the basis of their fluorescence spectrum, using array-based spectral detection. In our current research we continuously try to improve the robustness and ease-of-use of different advanced fluctuation methods, and continuously try to increase the number of fluorescence readouts (intensity, lifetime, anisotropy, spectrum) in routine imaging experiments.