Generation and exploitation of proton motive force: Biochemical and structural analysis of three bacterial integral membrane proteins.

Doktorsavhandling, 2007

Proton motive force, a necessity for all living cells, is generated and exploited by asignificant number of membrane-bound enzymes and transporters. The focus of thisthesis was to understand the physiological relevance, structure and mechanism of threesuch bacterial enzymes: proteorhodopsin; transhydrogenase; and AcrB. Proteorhodopsin, common in surface living bacteria, is demonstrated to exist also inphylum Bacteriodetes, the third most abundant class of ocean bacteria. Evidence is alsopresented that its presence stimulates growth of one such bacterium in the presence oflight. This finding indicates that proteorhodopsin associated growth enhancementcould contribute to the primary production in earth’s biosphere. Furthermore,optimisation of proteorhodopsin synthesis in vitro and from E. coli shows that yieldssufficient for structural studies by NMR and X-ray crystallography are achievable. Over-production of functional transhydrogenase with an altered sub-unit composition,where the β-subunit is split into two polypeptides, is presented. This construct wasutilised to confirm that transmembrane helix nine is adjacent to helices 13 and 14.Thus, it provides support for the suggestion that these three helices, presumablytogether with helix ten, form the proton translocation channel. An X-ray structure of intrinsic AcrB, the main cause of multi-drug resistance in gramnegativebacteria, is determined. Two novel structural features are observed: apreviously unsuspected transmembrane helix and a specific rotation of the periplasmicporter domain of AcrB. Using mass spectrometry the new helix is assigned asoriginating from a new subunit, the single transmembrane protein YajC. However,growth studies could not conclusively determine the functional role of YajC whenassociated with AcrB. Nevertheless, the observed twist might explain how TolC isopened to allow drug export from the cell, since this motion is consistent with thehypothesised "twists to open" mechanism for TolC activation.