- There is no shortage of challenges in modern science, including the task of obtaining high resolution images of biomolecular structures, such as proteins. Current atomic-resolution structure determination methods include x-ray crystallography and nuclear magnetic resonance (NMR), which present issues like protein crystallization and loss of resolution with increasing size. Dr. Wei Kong is working to overcome these challenges by developing a new atomic-resolution structure determination method called single-molecule serial electron diffraction imaging (SS-EDI). However, this method presents some challenges, because it will still expose samples to extreme conditions like high temperature, low pH, and different solvents. Understanding how proteins will react in these environments is important for determining conditions that will prevent proteins from denaturing or aggregating. Furthermore, observing a protein in native form gives insight into how the protein functions in biological conditions. Here, sfGFP is exposed to low pH and increasing concentrations of organic solvents to understand the level of stability the protein is able to maintain under the conditions of SS-EDI. Our findings suggest that sfGFP exhibits a conformational change below pH 5.4, though the process is reversible with recovery of initial pH. Additionally, sfGFP appears to aggregate in the presence of acetonitrile and the conformational effects of methanol remain undetermined.
Key Words: superfolder green fluorescent protein, single-molecule serial electron diffraction imaging, fluorescence, organic solvents, pH