Painfully Energetic : A tale of two proteins potentially connected

Sammanfattning: NADH:quinone oxidoreductase (Complex I) is the first enzyme of the respiratory chain and is involved in energy conservation generating an electro-chemical gradient across a membrane. The enzyme can be divided into a membrane spanning domain and a hydrophilic domain, which protrudes from the membrane. In the hydrophilic domain electrons from NADH oxidation are transported via a wire of iron-sulfur (Fe-S ) clusters to quinone, which is reduced. While the membrane domain is responsible for proton translocation to maintain the proton motive force, which is important for ATP synthesis. Large protein complexes like complex I have evolved from an assembly of discrete functional building blocks of which there are extant homologs. Two very different protein families, the Mrpantiporter and membrane bound [NiFe]-hydrogenases contain subunits which are homologous to complex I subunits. Part one of this work aimed to better understand the functional relationship between the related protein subunits of complex I, Mrp-antiporter and [NiFe]-hydrogenases. This knowledge will help us to elucidate the proton translocation pathway in complex I. First we compared the functional differentiation of complex I antiporter-like subunits with transporter subunits of the Hyc and Hyf hydrogenases and the 11-subunit complex I. For that we tested if the different subunits could rescue the growth of two salt sensitive Bacillus subtilis strain, which each lacked one of the two large Mrp-antiporter subunits (MrpA/MrpD). The 11-subunit complex I subunits could restore the growth in a similar manner as the complex I subunits, whereas the hydrogenase subunits could substitute equally well for the two MrpA and MrpD. We confirmed that 11-subunit complex I is a bona fide complex I. and that the hydrogenase subunits have intermediate forms of the antiporter-like subunits. Secondly we examined the functional relationship of the two homologous proteins MrpA from the Mrp-antiporter and NuoL from complex I. We located a stretch of amino acid residues which is conserved only in NuoL and MrpA, but not in the other complex I antiporter-like subunits or in MrpD. These residues were subjected to site directed mutagenesis and any resulting effects were examined in vivo by B. subtilis complementation studies and 23Na-NMR. Only one mutation (M258I/M225I) showed differences in the efficiency of cell growth and sodium efflux in both subunits, the other mutants were all able to cope with high salt levels.Ion channels are important for many processes in the cell and critically depend on gradients over membranes to execute their functions. They are involved in the detection of changes in the environment, which is an important survival mechanism for every organism. One of these ion channels is TRPA1, which belongs to the TRP superfamily of non-selective cation channels. TRPA1 can be activated by changes in temperature and voltage, as well as by a wide range of electrophilic and non-electrophilic chemicals. As structural information is limited, the exact activation mechanism is still elusive. The aim of the second part was to study the structural and functional changes of TRPA1 upon activation by temperature and chemical activators. We studied the effect of increased temperature and ligands on the conformation of mosquito TRPA1 (AgTRPA1), using intrinsic tryptophan fluorescence, SRCD and nanoDSF. We showed that the electrophilic ligands tested were quenching the tryptophan flourescence in the same way, suggesting a similar binding mechanism. We propose a putative model how temperature and ligand can activate AgTRPA1.Furthermore, we truncated the C-terminal region of human TRPA1, in an attempt to narrow down the minimal structural and functional unit of hTRPA1. This will facilitate future structural and functional studies of the activation mechanism.