| The major hurdle in the analysis and exploitation of complex membrane proteins is the overexpression of such proteins in a functional state. Although the mechanisms underlying synthesis, folding, membrane insertion and post-translational modification are largely known, the kinetics, dynamics and location of the individual steps of membrane protein biogenesis are poorly documented. We aim to determine the bottlenecks and roadblocks in membrane protein synthesis in order to obtain a mechanistic understanding of the membrane protein biogenesis pathway in Lactocococcus lactis. We will quantify the intermediates of membrane protein synthesis and determine their location and dynamics in the cell, using engineered and evolved strains that produce the proteins at improved levels and in a functionally competent state. We will use cutting edge single-molecule fluorescence methods (incl. super-resolution microscopy), state-of-the-art molecular biology (incl. deep sequencing of RNA), and biochemistry tools to unravel the intricacies of the membrane protein biogenesis pathway in L. lactis. |
