MEASURING HYDRODYNAMIC FORCE AND FRICTION ON LIPID ANCHORED PROTEINS

Lipid-anchored peripheral membrane proteins differ from transmembrane proteins in their freedom to explore the entire outer surface of a cell. Flows of surrounding fluid can rearrange these proteins by pushing them to the downstream cell edge, and this transport may be involved in flow mechanosensing. Previously, we developed an experimental method for measuring flow-induced lateral transport of neutravidin bound to biotinylated lipids in model membranes. Making general predictions about flow transport of proteins requires quantitative estimates of the hydrodynamic force and membrane drag. Here, we measure forces from shear flow directly by extending our method to compare flow mobility for lipid-anchored protein constructs with different sizes but identical lipid anchors and membrane compositions. To eliminate uncertainty in the number of biotinylated lipid anchors bound to each protein, we used monomeric streptavidin (mSA). We generated a series of protein constructs with increasing size: mSA, mSA-GFP, and mSA-MBP. We then compared the flow mobility of these constructs with that of commercially available streptavidin. The mobility of proteins measured by the experiment follows the expected order of mSA-MBP> mSA-GFP > mSA, correlated with their size. Our measurements of mobility and diffusion allow us to calculate the hydrodynamic force applied by flow, which is proportional to the hydrodynamic area of each protein. We compare our experimentally determined hydrodynamic areas with values obtained from molecular dynamics simulations. The hydrodynamic forces we apply to the protein constructs are on the order of piconewtons, similar to the forces applied by blood flow to membrane proteins on cells located at blood vessel walls. To demonstrate that flow-mediated protein transport can occur on living cells, we measure the flow-induced lateral transport of GFP-tagged plasma membrane proteins of different sizes on living COS-7 cells.