The major goals of this proposal are the determination, refinement and analysis of the dimer and monomer forms of Chromatium vinosum cytochrome c' (CCP) using the methods of protein crystallography. CCP undergoes a monomer-dimer transition upon binding a heme ligand, such as carbon monoxide, and as such is an ideal and tractable system for studying protein-protein association-dissociation and cooperativity in proteins. By looking at the specific conformational changes that promote the dimer vs. the monomer form, insight can be gained into the nature of protein binding in general. Molecular biology now has the tools to build or modify a protein of any sequence. However, knowledge is incomplete at the level of detailed molecular mechanisms for protein-ligand interaction, protein- protein assembly, and local conformational change, and this hampers intelligent design of artificial proteins specific for medical use. The proposed work will help bridge this gap by providing a detailed, high- resolution, crystallographic analysis of a protein-protein association- dissociation system. Ideally, four crystallographic structures will be determined: the dimer structure of CCP in its reduced and oxidized forms, and the monomer structure with ligand bound and unbound. Crystals which diffract to 1.9 A have been grown of the dimer and data collected on both the oxidized and reduced forms. Determination of these two structures will be the first priority and will give much information about the ligand binding site and the dimer interface. Three heavy-atom derivatives have been identified of the oxidized dimer and data has been collected. The known sequence of CCP will be used to interpret electron density maps and the structures will be refined. Analysis will focus attention on the dimer interface and the heme environment, with particular attention paid to the movements of side-chains and how these affect the interaction between the two protein molecules. Specifically of interest is the heme and the influence of ligand upon the heme conformation. Analysis of the conformational changes will be assisted by comparison with the existing structure of Rhodospirillum molischianum cytochrome c' which does not exhibit dissociation. The monomeric form has been produced and crystallization trials are underway in our anaerobic chamber. Crystals of this form will be of value in further analysis of the system, however, alternatives using other techniques making use of the dimer coordinates are planned if crystals are not forthcoming soon.