Research Summary

Molecular electron microscopy (EM) is a versatile tool to analyze the structure of molecules that are not easily amenable to structure determination by the more established techniques of X-ray crystallography and NMR spectroscopy. Our group studies the structure of macromolecular complexes and integral membrane proteins using both single particle EM and electron crystallography of two-dimensional crystals.

We are interested in visualizing interactions between proteins that lead to cell adhesion or the formation of membrane junctions. We use ovine lens fiber cells as a system to visualize protein-mediated membrane junctions. Our two main projects concern the lens-specific aquaporin AQP0 and the lens-specific tetraspanin MP20. Whereas AQP0 mediates membrane adhesion via direct interactions of AQP0 tetramers in juxtaposed membranes, MP20 is likely to attach membranes to each other through interactions with galectin-3, a known cell adhesion modulator. Our work on AQP0 also led us to systematically study the non-specific interations of lipids with membrane proteins. In collaboration with Tim Springer, we also study integrins, focusing mainly on their activation and ligand binding mechanisms.

A second focus of our group is proteins that mediate iron transport across membranes. We have begun by visualizing the complex formed by iron-loaded transferrin (Tf) with its receptor (TfR), which delivers iron to cells by endocytosis. We have expanded this work to the hemopexin-hemopexin receptor complex, which delivers heme to cells in a way analogous to the iron delivery by the Tf-TfR complex. In addition, we have begun work on the structure of transmembrane iron transporters.

We also study the structure of macromolecular machines. In particular, we are interested in chromatin remodeling complexes. Our goal is to define the structural changes in DNA packing that occur upon modification of nucleosomes by remodeling complexes, such as the SIR and RENT silencing complexes (in collaboration with the Moazed group), the RSC complex, and Polycomb group protein complexes (in collaboration with the Kingston group).

As we are working on fundamental biological questions, we strive to maximize the repertoire of our facility to take advantage of the full potential of molecular EM. We are thus also working on advancing the methods used in molecular EM. Examples are the adaptation of X-ray crystallographic approaches to electron crystallography and the adaptation of the monolayer 2D crystallization approach to single particle EM analyses, which led to the development of the “Monolayer Purification” technique and the “Affinity Grid”.

Selected Publications:

Hite, R. K., Gonen, T., Harrison, S. C., and Walz, T. (2008). Interactions of lipids with aquaporin-0 and other membrane proteins. Pflugers Arch. 456: 651-661.

Kelly, D. F., Dukovski, D., and Walz, T. (2008). Monolayer purification: a rapid method for isolating protein complexes for single-particle electron microscopy. Proc. Natl. Acad. Sci. USA 105: 4703-4708.

Skiniotis, G., Moazed, D., and Walz, T. (2007). Acetylated histone tail peptides induce structural rearrangements in the RSC chromatin remodeling complex. J. Biol. Chem. 282: 20804-20808.