The three-dimensional structures of oxygenated and deoxygenated forms of Limulus subunit II hemocyanin have clarified the molecular controls of function in these large oxygen-transport proteins. The native hemocyanin of Limulus consists of 48 subunits with eight types of polypeptide chains. Each subunit type is capable of reversibly binding oxygen at a dicopper active site. The subunits differ in primary structure, ligand affinities, and sensitivity to assorted allosteric effectors. This study is designed to test mechanistic ideas using a combination of biochemical, biophysical and molecular biological techniques. Hemocyanin structures will serve as prototypes for a large class of multisubunit assemblies that require multiple components for complete function. Comparison of the structure of Limulus II hemocyanin with preliminary structural results for subunits IIIa and IV suggests the mechanism for subunit II cooperativity may well be general for all arthropod-type hemocyanin subunits. cDNA has been isolated from a Limulus library that codes for a 24 residue peptide identical in sequence to part of subunit a from a southeast Asian horseshoe crab. Once the nucleic acid sequences for the subunits are available, construction of designed variants and smaller reversible oxygen-binding entities will be pursued. The structure of subunit IIIa is incomplete, providing further insight into the chemistry of the dicopper active site. Protons, divalent cations and specific anions act as modulators of oxygen affinity in Limulus hemocyanin.
2. Non-techincal
Oxygen transport from the environment to the tissues is vital for the survival of multicellular organisms. In humans, this function is carried out by the protein hemoglobin, the red pigment in blood. In some arthropods such as lobsters, crabs, crayfish and horseshoe crabs, the oxygen transport protein is hemocyanin ("blue blood"). The hemocyanin of the horseshoe crab is about 3.3 x 106 Daltons and is made up of 48 subunits of eight different types. All of the subunit types can reversibly bind one molecule of oxygen at their dicopper active sites. When the subunits are assembled into the 48mer, they bind oxygen cooperatively--once the first molecule of oxygen is bound it becomes easier to bind subsequent molecules and once the first molecule of oxygen is released at the tissues, later ones come off more readily. The cooperative binding of oxygen is regulated by allosteric effectors such as chloride and calcium ions. The goal of this project is to understand in molecular detail how this complex protein assembly carries out its physiological functions. The three dimensional structures of all eight subunits and their complexes are being determined using the methods of x-ray crystallographic analysis. Study of structures of hemocyanin subunits with and without oxygen bound suggested which amino acids are key in effecting cooperative, reversible ligand binding. By using the techniques of molecular biology, the proposed key amino acid residues can be changed and the altered structures physical properties studied to test and extend the mechanism. The structures of various forms of the whole hemocyanin molecules will also serve as a prototype for larger more complicated structural assemblies.
