Department of Biology
Research Opportunities
Dr. Robert H. Kretsinger
Protein Structure, Function, and Evolution
In collaboration with Carl Creutz in Pharmacology we have determined the crystal structure of the calcium bound form of annexin VI to 2.9 Å resolution. The structure consists of two discs (domains 1-4 and 5-8) that are tipped 90o to one another. Under some conditions the two discs lie on a lipid monolayer both in the same orientation; under other conditions disc 5-8 is flipped over to antiparallel, as seen in 3D reconstructions from electron micrographs of 2D crystals. The crystal structure of T356D, at 2.5 Å, intended to mimic phosphorylation, reveals five Ca2+ ions not found in the wild type annexin VI.
In collaboration with Ron Bauerle, Department of Biology, we have determined several crystal structures of the phenylalanine inhibited form of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from E. coli. DAHPS condenses erythrose-4-phosphate and phosphoenolpyruvate to form DAHP, the precursor of all aromatic compounds in bacteria and plants. The sites of E4P, Mn2+, and PEP are located near the C-end of a (b/a)8 TIM-barrel. We have determined the structure of DAHPS(Phe)*Mn*PEP with and without the feedback inhibitor, phenylalanine, bound and have traced the changes in conformation from the Phe site to the active site. E4P can no longer bind and the PEP flips its orientation in DAHPS upon binding of Phe 20 Å away! Numerous mutants of DAHPS have been analyzed in terms of these crystals structures. We have proposed a mechanism of enzyme action and a mechanism for feedback inhibition.
With Julie Sando, Anesthesiology, we have made 2D reconstructions from electron micrographs of protein kinase C-d, of its regulatory domain, and of PKCd complexed with myelin basic protein. The C1 domain of RDd is imbedded in the phospholipid monolayer. The binding of MBP to the catalytic domain of PKCd causes a shift in orientation of the catalytic domain relative to the regulatory domain. These E.M. studies are being extended to 3D. We anticipate being able to fit existing crystal structures of domains of PKC into the low resolution 3D model of the intact protein interacting with a membrane surface. Our lab has a long standing interest in protein evolution; we have focussed on generating and interpreting dendrograms of EF-hand containing proteins as well as those with leucine rich repeats. We have promising results on a new program whose goal is ab initio prediction of protein structure.