We have cloned the tolQ and tolR genes from various Gram-negative bacteria into several expression vectors. The TolQ protein construct contains a C-terminal histidine tag while the TolR protein contains an N-terminal strep tag. Complementation and physiological tests with tolQ and tolR deletion mutants result in a restored wild-type phenotype and the tags do not interfere with function. However, expression levels have not yet been optimized for structural studies. Future work includes cloning tolQ, tolR and tolA into expression vectors using different promoters to try to obtained functional, membrane-inserted protein in good yields. We have initiated small scale purification experiments with the tolQR construct using a Ni affinity column. Our preliminary results show that when TolQ and TolR are overexpressed, they form a complex with chromosomally expressed TolA, resulting in small yields of the triple complex. Over the past year, we have isolated individual proteins and complexes, finding that TolQR is less susceptible to degradation than when purifiying Tol Q or TolR alone. Yields of purified protein are currently not sufficient for crystallization experiments so we will continue to optimize expression. We are also studying expression systems for ExbB (a TolQ homolog) and have obtained an expression system that produces large quantities of membrane-inserted protein suitable for structural studies. Initial purification experiments show two major proteolytic fragments which are under analysis currently. We intend to correct proteolysis by deletion or substitution mutagenesis and then proceed with purification and crystallization experiments on ExbB. This system produces sufficient protein levels for crystallization and structural studies are ongoing. Structures of TolQ, TolQ-R, TolQ-R-A (and Ton complex as well) are important for knowledge of the assembly of the various Tol complexes and their effect on membrane integrity, but they will also be valuable for understanding the energy transduction mechanism communicated over a substantial distance through the periplasm.
