Membrane proteins perform vital functions like respiration, signaling and nutrient uptake. Complex formation between two or more membrane proteins is limited by their diffusion coefficient in the plane of the membrane. Despite a lot of work on model membranes, little is known about lateral diffusion of proteins in prokaryotic membranes.
We used single particle tracking (SPT) to analyze the lateral mobility of trans-membrane proteins of varying size and function fused with green fluorescent protein in E. coli using single-molecule wide-field epi- fluorescence microscopy. Diffusion coefficients obtained for these proteins from mean squared displacement and cumulative probability distribution analysis show a weak decrease in mobility with increasing trans-membrane inclusion radius, essentially as predicted by theoretical models and in vitro studies. However, the measured diffusion coefficients in vivo are at least 30 fold lower than in vitro. This is due to the large number of proteins embedded in the bacterial membrane compared to the lipid bilayers used for in vitro studies. This crowding effect might significantly affect membrane-protein activity and function, for example signal transduction and transport processes in bacteria.
Furthermore, we have developed a method to correct for the deformation of diffusion trajectories on the curved membrane of bacteria, which occurs as a result of projecting the trajectory onto the plane of the camera. Our method, called inverse projection of displacement distributions (ipodd) is able to correct for this projection artefact, and enables us to obtain correct diffusion coefficients and, more importantly, obtain correct distributions of displacements. Those distributions contain information about whether the diffusion is homogeneous, or whether the diffusing molecules are heterogeneous due to for example different environments in the membrane or complex formation with other proteins. We have recently started to apply the developed methodology to study complex formation in protein translocation.