The elucidation of the structure and function of cellular membrane components remains today one of Science's grand challenges. The challenge becomes immediate as we seek to understand the molecular basis of such common lethal genetic diseases as cystic fibrosis where the defect is attributed to a malfunctioning transmembranal chloride channel. How is the channel built into the membrane? Does the membrane influence channel activity? What critical components does it interact with in, on, and next to the membrane? These are crucial questions concerning membrane protein topology that remain unanswered for want of appropriate methods and model systems. With these long term objectives in mind, we propose to tackle particular aspects of the membrane topology challenge by introducing a new experimental approach. This will be used to determine the precise disposition of cytochrome c, a component of the electron transport system, at the membrane surface. While the crystal structure of the protein is known, the manner in which cytochrome c docks on the membrane surface and how it interacts functionally with its partners in the electron transport system is still in question. X-ray standing waves (XSW) will be used for the first time to determine, with subangstrom resolution, the position of the intrinsic heavy atom, iron, In a cytochrome c monolayer electrostatically bound to a Langmuir-Blodgett lipid film deposited on a solid support. The latter will be used to generate the XSWs. Protein orientation will be confirmed by performing additional XSW measurements to determine i) selenium atom location in cytochrome c modified at three or more sites with selenomethionine substituting for the native methionine and ii) ruthenium atom location in cytochrome c derivatized singly at Cysl02 on the protein surface. In this way, the location of at least five known reference points in the protein above the membrane surface will be determined. This, together with the known structure of the protein, will be used to establish unequivocally the orientation of cytochrome c at the membrane surface. Changes in orientation upon reduction and oxidation will be determined and correlated with conformational changes seen in the crystal structure. Additionally, the distribution of the protein in the aqueous phase next to the membrane and how this is modulated by the membrane lipid characteristics as well as the ionic composition of the membrane bathing solution will be determined and used to test the hypothesis that at least two pools of cytochrome c exist in mitochondria. Separate measurements will make use of the apocytochrome c and cytochrome c covalently bound to the membrane surface. A successful determination of the membrane docking mechanism for cytochrome c and the manner in which the protein distributes between membrane-bound and soluble forms paves the way for the elucidation by the XSW technique of the structure and disposition of a host of integral and peripheral proteins whose membrane properties remain elusive for lack of appropriate methodologies.
