- Obtaining high-resolution structures of biological macromolecules has become one of the biggest challenges in the scientific world. Dr. Wei Kong’s lab is currently working to overcome the limitations of current structure resolution techniques, such as x-ray crystallography and nuclear magnetic resonance spectroscopy. Kong’s group hopes to develop a new protein imaging method, called single-molecule serial electron diffraction imaging, that can be applied to determine protein structures without the need for crystallization and with no limitation on protein sizes. However, this protein imaging technique, like many other analytical techniques, requires exposing protein samples to unideal, non-native conditions. Understanding how these types of environments can alter protein conformations is important for determining the conditions that will optimize the reliability of the protein structure model that is obtained. Here, fluorescence measurements of super folder green fluorescent protein—an ultra-stable strain of green fluorescent protein engineered in 2006—were taken under extreme temperature and organic solvent conditions in order to assess its level of denaturation and aggregation within the conditions of single-molecule serial electron diffraction imaging. Our findings suggest that super folder green fluorescent protein tends to aggregate in the presence of acetonitrile and denature in the presence of methanol. The protein is also capable of complete renaturation after exposure to temperatures up to 80ºC, and it retains 90% of its fluorescence in both organic solvent conditions at 80ºC for ≥4 s. This information is valuable for Kong’s group as they continue to develop their protein imaging method and also provides insight into the structural equilibria of proteins when placed in non-native conditions.