Mammalian fatty acid synthase (FAS) is a multifunctional polypetide that includes all catalytic activities required for biosynthesis of long-chain fatty acids. Whereas the biochemistry of the system is well understood, structural analysis of FAS has been hindered by the size (MW 540 kDa) and complexity of the enzyme. The functional form of FAS is a homodimer including two polypetides, each comprising six catalytic activities and a phosphopanteteine prosthetic moeity involved in translocating substrates from one active site to the next. Not surprisingly, FAS presents a very large degree of conformational variability. Because of its biological relevance, complexity, and dynamic behavior, FAS constitutes a target of great interest for EM studies. Our objective is to understand its molecular organization, and to obtain information about active site interactions and conformational changes required for catalysis.

Besides the relatively large MW of animal FAS, a considerable hurdle for its structural characterization has been the large conformational variability of this multifunctional protein.

The use of paused mutants allowed us to reduce the conformational variability of FAS particles, which in turn made possible calculation of a 3-D reconstruction from images of particles preserved in amorphous ice at a higher resolution. Analysis of the volume reveals that its symmetry is not consistent with a fully-extended, anti-parallel arrangement of two FAS monomers.

To further investigate the organization of the functional FAS dimer, the structure of an FAS monomer was determined.

FAS monomers were obtained by cold-induced dissociation of FAS dimers, preserved in stain, and imaged. A reconstruction calculated using the random conical tilt method reveals that an FAS monomer has a coiled conformation. It appears likely that the monomer conformation is largely conserved in the FAS dimer, which might be formed by two overlapping, intertwined monomers.

The FAS monomer N-termini were labeled with a gold cluster thorugh properly engineered 6xhistidine tags. The N-termini were localized to the central portion of the FAS structure. Chemical crosslinking studies indicate that the N-termini are separated by a very short distance (less than 1 nm).

Our long-term objective in this project is to develop a dynamic understanding of FAS, which can be considered as a true "molecular fatty acid assembly line". By studying the structure of FAS monomers and dimers we will understand structural conformations changes induced by dimerization that are essential for catalysis. A combination of X-ray crystallography and EM studies will eventually result in localization of all active domains. Obtaining information about the conformational changes that are necessary for catalysis is a challenge that can almost uniquely be met by macromolecular EM. We will determine the structures of a number of paused mutants and correlate them to their biochemical and functional profiles to obtain a series of "snapshots" that will reveal the workings of FAS as it proceeds along the fatty acid synthesis cycle.