Proteins are known to adopt specific (but dynamic) three-dimensional structures that allow them to carry out their function. It is well recognized that this process doesn’t occur by a random search but is instead a biased search. We are interested in understanding the factors that guide the protein to the correct structure or set of structures.
Protein misfolding and aggregation have been implicated in a number of human diseases. One of the proteins that we study, human superoxide dismutase (hSOD1), has been implicated in amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease. As part of a collaborative effort with Jill Zitzewitz and Bob Matthews, we have been studying the
Live cell microscopy: The other question that we are interested in addressing focuses on understanding the factors leading to toxicity in live cells. To this end. we have built a 2-photon (also capable of 1-photon confocal) laser scanning microscope custom suited for long-time live cell studies. The goal is to track the spatial localization and oligomerization of SOD1 in live cells and correlate that with toxicity.
Another area that we are interested in is using single-molecule fluorescence methods for studying protein folding reactions, protein-protein interactions and protein oligomer formation. Single-molecule methods can be used to probe protein oligomer formation and also, using fluorescence correlation spectroscopy, probe dynamics from the sub-microsecond to tens of seconds timescale. To enable these studies we have built
If the hypothesis that the gain-of-function toxicity of SOD1 is triggered by a preferential increase in unfolded state population is correct, than one approach to mitigating the destabilization effect of disease-related mutations or zinc deficiency might be to use a small-molecule to preferentially stability the native state.
Another part of this study is focused on identifying the small oligomers present and the regions of SOD1 involved in stabilizing these small oligomers. We are pursuing these studies by a number of biophysical tools, including hydrogen exchange mass spec and fast photochemical oxidation (in collaboration with Michael Gross’ lab at Washington University in St. Louis), single-molecule methods and electron microscopy.