Resumen de Investigation of the phospholipid peripheral region of lactose permease in model membranes
María Carmen Suárez Germà
The interaction between a membrane protein and its surrounding phospholipids is thought to be crucial for the correct folding and function of the protein. This thesis is focused on the investigation of the interplay between Lactose permease (LacY), a paradigm for secondary transporters present in the inner membrane of Escherichia coli and model systems mimicking its natural lipid environment. Since the role of phospholipids in LacY’s activity is currently being refined, this work represents a contribution to the field by studying the interaction at two different levels: (i) the LacY interplay with the phospholipids present at the annular region in the vicinity of the protein was studied through FRET measurements between a single-tryptophan LacY mutant and diverse pyrene-marked phospholipids, and (ii) the LacY interaction with the more distanced bulk phospholipids was studied through supported proteo-lipid sheets that were analysed using topography, force-spectroscopy and force-volume Atomic Force Microscopy modes. First, after validating LacY preference for phospholipid fluid (L?) phases in the studied two-component model systems, a different composition between bulk and annular regions was confirmed. Hence, bulk lipids, which were assimilated to the phospholipids in L? phase, were mainly formed by PG, while PE was the main component of the annular region. This points to a direct annular phospholipid-LacY selectivity because it discards a random phospholipid distribution near the protein. Second, the LacY selectivity for precise phospholipid species at the annular region was found to be related to: (i) a neutral charged phosholipid (PE or PC, with preference for the former), and (ii) phospholipids with large negative spontaneous curvature (C0) (DOPE > POPE). In addition, D68 was revealed as an important amino acid for the protein annular lipid selectivity. Third, the interaction between LacY and the bulk lipids was described as reciprocal. Accordingly, the presence of the protein largely modified the topography and the nanomechanics of the lipid system, especially for the L? phase, whilst the nanomechanics of LacY itself were different depending on the surrounding lipid matrix: more force was needed to pull LacY form the DPPE:POPG (3:1, mol/mol) system than from the POPE:POPG (3:1, mol/mol) one. Therefore, the bilayer lipid composition seems to determine the forces governing the LacY tight interaction with the membrane and can be thus decisive for the protein correct insertion and activity.
