Structure-function studies on the hetero-oligomeric cytochrome b6 f complex have led to successful crystallization and initial characterization of well-diffracting crystals of the complex from the thermophilic cyanobacterium, Mastigocladus laminosus, whose structure analysis is underway. They have also led to a new "lipid enrichment" methodology for the crystallization of such integral membrane proteins, considering the protein in detergent micelles as a protein-detergent-lipid complex. The presence and the masses of eight subunits of the complex from M. laminosus and nine subunits from the higher plant complex, including the newly discovered FNR component, have been characterized by mass spectroscopy. The MS analysis removed uncertainty as to exactly how many small polypeptides are present in the complex. A unique aspect of the role of lipid in the function and structural stabilization of the complex arose through the discovery of one equivalent of lipid-like beta-carotene and chlorophyll alpha in the complex. The lipid parameters of the "enrichment: methodology will be examined. It is proposed to test the applicability of the new lipid enrichment methodology to the b6f complex in a transformable cyanobacterium and four other unrelated integral membrane protein systems. The potential application of this methodology is important for studies on integral membrane proteins, as there are at present only 23 solved independent IMP structures,and 56 in total. This creates a limiting step in proteomic analysis since 20-30 % of gene products are membrane proteins. The function of the Rieske iron-sulfur protein (ISP), which has been proposed to undergo a unique large amplitude rotation-translation in its electron transfer function, was analyzed through: (i) the viscosity dependence of the electron transfer function, (ii) stopped-flow kinetics of diffusion limited delectron transfer in solution, and (iii) mutagenesis analysis of the linker region, the "hinge of the proposed rotation. The ISP motion is constrained in vivo. The motion does not depend sensitively on the structure of the linker region, but the linker has a minimum length of 7 residues. It is proposed to directly measure the kinetics of motion of the ISP soluble domain triggered by electron transfer.
NIH ExPorter
NIH ExPorter