9305250 Magnus This project will employ x-ray diffraction methods to establish the three-dimensional structures of an important class of biological macromolecules known as hemocyanins ("blue bloods"). Proteins that bind and carry oxygen are of fundamental importance to multicellular animals. Hemocyanins are of particular interest because they are larger and more complex aggregates of subunits than have routinely been studied previously and because of the interest in the chemical nature of the interaction between copper and oxygen. The structures of hemocyanins, copper-containing oxygen-transport molecules found in a variety of invertebrates including shrimps, lobsters, crabs, scorpions and spiders, show the oxygen binding geometry in atomic detail. Because the data collection time for an x-ray diffraction experiment usually spans several days, the actual mechanistic steps involved in binding, which occurs on a millisecond time scale, are not observed directly. Instead by studying the protein structure in the presence and absence of oxygen, it is possible to deduce which the 628 amino acid residues that comprise the hemocyanin protein subunit are likely to be involved in oxygen binding and its regulation. These predictions may be tested by using molecular biological techniques to produce genetically tailored versions of hemocyanin whose properties may also be examined crystallographically and spectroscopically. %%% The goal of the proposed project is to extend understanding of how protein structure effects control of functions in hemocyanins. These copper-containing proteins are responsible for facilitating oxygen uptake and delivery in a variety of invertebrates. Cooperative ligand binding in the large molecular weight subunit aggregates that comprise hemocyanin is modulated by an assortment of effectors including protons, divalent cations and chloride ions. The proposed x-ray crystallographic studies of the horseshoe crab, Limulus polyphemus, will prov ide information of the conformations of high and low affinity forms of hemocyanin, leading to a better picture of how cooperative ligan binding occurs. Complementary functional studies will be performed using spectroscopic and kinetic measurements to characterize hemocyanin fragment, subunits, aggregates and chemically modified forms. ***
