The work plan comprises 6 tasks with a reasonable timeline, designed to ensure that the objectives are successfully reached:
Task 1
Task 1 will be dedicated to developing expression systems for DsrMKJOP from two different organisms, optimizing expression and purification conditions, and producing the WT recombinant Dsr complex.
Task 1
Task 1 will be dedicated to developing expression systems for DsrMKJOP from two different organisms, optimizing expression and purification conditions, and producing the WT recombinant Dsr complex.
Task 2
Task 2 will be dedicated to producing variants of the Dsr complex, and also in vivo growth studies of d. vulgaris strains expressing these variants.
Task 2
Task 2 will be dedicated to producing variants of the Dsr complex, and also in vivo growth studies of d. vulgaris strains expressing these variants.
Task 3
Task 3 will involve molecular and catalytic characterization of the recombinant Dsr complex and variants, and functional studies in liposomes to assess proton translocation.
Task 3
Task 3 will involve molecular and catalytic characterization of the recombinant Dsr complex and variants, and functional studies in liposomes to assess proton translocation.
Task 4
Task 4 will involve Cryo-EM structural studies of the Dsr complex in different redox states and interacting with substrates, and EPR and MCD spectroscopic studies to investigate the mechanism and properties of cofactors.
Task 4
Task 4 will involve Cryo-EM structural studies of the Dsr complex in different redox states and interacting with substrates, and EPR and MCD spectroscopic studies to investigate the mechanism and properties of cofactors.
Task 5
Task 5 will involve inhibition assays with the Dsr complex screening a range of known inhibitors of quinone binding sites, and other commercially available compounds based on molecular docking studies and in silico screening. The best inhibitors will be tested in vivo with two gut bacteria.
Task 5
Task 5 will involve inhibition assays with the Dsr complex screening a range of known inhibitors of quinone binding sites, and other commercially available compounds based on molecular docking studies and in silico screening. The best inhibitors will be tested in vivo with two gut bacteria.
The methods used will include molecular biology and genetic manipulation of D. vulgaris to generate the variants, microbial growth studies, protein purification and characterization, enzymatic and inhibition assays through spectrophotometry, functional assays in liposomes with stopped-flow experiments, Cryo-EM structure determination, EPR and MCD spectroscopies, molecular modelling and docking studies, and general microbiological, biochemical and molecular biology techniques.