Grant: Research Tools and Instruments - Category 1 (<$150,000)

My research program is focused on the membrane protein complexes of the respiratory electron transfer chain, and the accessory proteins that assist in the assembly of these complexes. Our present focus is on the enzyme complexes of the cytochrome oxidase family. We are interested in aspects of the cytochrome oxidase mechanism that results in the consumption of oxygen and storage of the resulting free energy that can be used for other cellular work. There are accessory proteins that help to construct proteins such as the cytochrome oxidase complex. Cytochrome oxidase consists of up to 13 protein subunits and these subunits have associated with them two iron ions and three copper ions. The metal ions play a direct role in the reactions catalyzed by cytochrome oxidase. In such metalloproteins the properties of the metal ions are modulated by the protein matrix and our approach to understand this subtle interplay begins with the purified protein. We use the purified oxidase to look at aspects of its reaction with oxygen, reductants and how these reactions are modulated by the interactions with membrane lipids. We are also exploring how the metal centers of the oxidase are assembled, and the protein molecules that are involved in this process. Each of our projects on the oxidase directly, or on the accessory proteins involved in its assembly begins with the purification of the protein from its biological source. These purifications involve a series of controlled steps that take place over 1-2 days, or up to one week in time. Our work relies heavily on the preparation of these proteins in large amounts in a highly purified state. Such demands require careful and controlled conditions to achieve reproducible, highly purified products in high yield. This outcome is most readily achieved through use of an automated chromatography system that affords the user a high degree of control over the purification process. The equipment requested here will allow personnel in my group to obtain the highly purified proteins that are a prerequisite for their detailed studies. In addition the use of the requested equipment supplies valuable training experience for technicians, undergraduate and graduate students in the fields of biochemistry and biophysics.