The objective of this project is to elucidate atomic-level principles by which cooperative protein function depends upon the stereochemical details of assembly. We will use three excellent model systems from the invertebrate hemoglobins to explore this problem. These molecules range from a simple dimeric hemoglobin with strong cooperativity but no environmental modulation to a virus-sized aggregate with significant regulatory features. The central role of cooperativity in biological function suggests that principles obtained from such a study will have wide ranging applications to many other physiologically important systems. A powerful combination of structural, functional and genetic approaches will be used to probe the cooperative mechanism in the simple Scapharca dimeric hemoglobin. This molecule, with two communicating oxygen binding sites, represents the simplest possible cooperative system. Crystals of Scapharca dimeric hemoglobin diffract to very high resolution, making it an excellent model for exploring atomic-level principles of cooperativity. We will probe the role of individual amino acid residues in the cooperative mechanism by site-directed mutagenesis, then analyze the resulting molecules by x-ray crystallography and thermodynamic measurements of dimer assembly. The globin-folded building block found in Scapharca dimeric hemoglobin (and in human hemoglobin) is also used in the assembly of Scapharca tetrameric hemoglobin and the 3.9x10(6) Dalton Lumbricus hemoglobin. The assembly of these larger molecules leads to additional regulatory features. We will use x-ray crystallographic analysis to determine the structural basis for cooperativity and regulation of these molecules. These structures may hold important secrets about how modulation of interfaces can be achieved within a given protein fold.