This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In collaboration with ACERT we wish to study the interactions between a key human complement regulator (DAF;CD55) and (i) bacterial adhesins e.g. the DraE of enteropathogenic Eschericia coli or (ii) the human T-cell co-stimulatory molecule, CD97 using DEER and DQC. For all three components we have high resolution X-ray crystallographic structures and also have chemical shift mapping data for the CD55 binding site on both ligands. We now seek to derive constraints from DEER and DQC (ACERT) that will allow us to dock the complexes together. Our CD55 construct is already engineered with a free Cys at the C-terminus and we have successfully used this to investigate another CD55-ligand pair using DEER. However, the combined application of our DEER technology with the DQC methodologies available at ACERT allows us to determine distance distributions for a larger range of distances at a much improved S/N, hence, providing superior data for protein docking. All three proteins are expressed in E. coli. The CD55 and CD97 are refolded post-expression to allow correct formation of their many disulphide bonds. The DraE is not refolded but also contains a single disulphide bond. It is the presence of natural disulphide bonds that makes this application adventurous as it is difficult to predict whether it will be possible to generate functional, correctly folded proteins, with additional Cys inserted. Our strategy will be to use the atomic structures to identify surface exposed residues (e.g. Ser) where mutation to Cys is unlikely to disrupt the structure (once folded). Once proteins are characterized by DEER and other cw and pulsed EPR technique here in Oxford they will be investigated further by the DQC methodology developed in the Freed laboratory yielding distances between the two spin-labeled proteins from as short as 10 ? and to as long as 90 ?. In all experiments, we plan the use of deuterated spin labels as well as fully deuterated buffer solutions.
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