Collaborative work between members of the Pielak Group, Nancy Thompson, and members from the departments of Biochemistry and Biophysics, Cell and Molecular Physiology, published in Biochemistry describes how fluorescence recovery after photobleaching was used to measure the diffusion coefficient of green fluorescent protein (GFP, 27 kDa) in Escherichia coli in the presence or absence of four coexpressed proteins: cytoplasmic maltose binding protein (42 kDa), tau-40 (45 kDa), α-synuclein (14 kDa), or calmodulin (17 kDa).
The GFP diffusion coefficient remains constant regardless of the type of coexpresseed protein and whether or not the coexpressed protein was induced. The researchers conclude that expression of these soluble proteins has little to no effect on the diffusion of GFP. These results have implications for the utility of in-cell nuclear magnetic resonance spectroscopy.
Translational diffusion is central to most biological processes especially in prokaryotic cells. Since these cells lack the motor proteins and developed cytoskeletal networks of higher organisms, diffusion is often their primary source of intracellular movement. Measuring diffusion in micron-sized structures, however, is challenging because of their small size compared to classical optical resolution.
The Thompson and Pielak groups have collaborated in developing a new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching to overcome these obstacles.