Absolute measurement of phosphorylation levels in a biological membrane using atomic force microscopy: the creation of phosphorylation maps

We show that it is possible to produce phosphorylation difference maps of biological membranes under conditions which reflect those in vivo and in which proteins remain functional. We also demonstrate that absolute levels of phosphorylation are retrieved through the application of an appropriate calibration method. Finally we show that the kinetics of phosphorylation/dephosphorylation can also be monitored. These methods are demonstrated on photosynthetic membranes from higher plants, for which protein phosphorylation is the dominant regulatory mechanism. We show directly that the most recent estimates of the phosphorylation levels in this system are reasonably accurate. Phosphorylation difference maps show that the distribution of phosphates is not even, with significantly higher levels at the membrane margins and patches of high phosphate density next to patches of low charge density.