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Electron Microscopy of Membrane Proteins and Chromatin Remodeling Complexes
Summary: Thomas Walz uses molecular electron microscopy to study how membrane proteins interact with lipids, how membrane channels conduct specific solutes, and how protein complexes can change chromatin structure.
Molecular electron microscopy (EM) can address questions that are not accessible to any other structural technique. We strive to take full advantage of the unique opportunities provided by molecular EM to study structural aspects of fundamental biological questions.
Structural Studies of Membrane Proteins in Their Native Environment
X-ray crystallography of three-dimensional (3D) crystals of detergent-solubilized membrane proteins is a powerful approach for obtaining high-resolution structural information on membrane proteins. Ideally, however, membrane proteins should be studied in their native environment, the lipid bilayer. We therefore reconstitute membrane proteins with lipids into two-dimensional (2D) crystals, which we then study by electron crystallography.
Understanding lipid-protein interactions: the aquaporin-0 membrane junction. Direct structural information on the conformation of lipids in bilayers and their interactions with proteins has been hard to obtain. Much of our knowledge derives from crystal structures of membrane proteins with specifically bound lipid molecules. Our electron crystallographic analysis of aquaporin-0 (AQP0) reconstituted with the lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) into double-layered 2D crystals at 1.9-Å resolution revealed nine DMPC molecules (see the figure). Since AQP0 has no specific lipid binding sites and DMPC is not a native lipid, our structure shows a nonspecific mode of protein-lipid interactions.
AQP0 forms 2D crystals with several lipids, offering an unusual opportunity to ask fundamental questions about how lipids interact with membrane proteins. By visualizing the structure of AQP0 2D crystals obtained with lipids with different acyl chains and different headgroups, we hope to learn how the bilayer structure adapts to accommodate the hydrophobic belt of membrane proteins and which lipid characteristics are most important for interactions with membrane proteins.
Understanding channel specificity: urea and iron transporters. Channel specificity is a crucial determinant of the internal milieu of a cell. Often the same solute is transported by members of different membrane protein families. We aim to understand how membrane proteins with different folds create channels with related or identical specificities. To address this question, we are studying membrane transporters for urea and iron.
Urea must be transported into and out of a variety of cells, for which a number of mechanisms have evolved. We pursue structural and functional studies of individual members of two groups of urea channels: the aquaporin (AQP) and urea/amide channel (UAC) families. Although both conduct water and urea, they share no structural homology. AQP9 has much broader substrate specificity than any other aquaporin. It is permeable to water, glycerol, and urea, as well as to polyols, carbamides, purines, pyrimidines, nucleosides, monocarboxylates, and even arsenite. One intriguing question is how AQP9 can conduct large neutral solutes while still being impermeable to protons. To answer this question, we will attempt to visualize solutes in the channel by soaking and co-crystallization experiments. We will further explore solute conduction by AQP9 and its broad substrate specificity by molecular dynamics simulations and mutational analysis of key residues in the channel. Almost nothing is known about the structure of members of the UAC family. To elucidate the structural basis for urea transport and channel specificity in UAC channels, we will determine the functional characteristics of these channels by stopped-flow fluorimetry, using purified proteins reconstituted into liposomes, and work toward characterizing their structures by electron crystallography of 2D crystals.
The redox-active metal iron is a vital nutrient for living cells. We are interested in proteins that mediate transmembrane iron transport and, in particular, how different transporter families accomplish specificity for iron. To address this question we are working on the structural characterization of members of the Nramp family of iron importers and of mammalian ferroportins, the only known iron exporters. We are again interested in how Nramp proteins and ferroportins have evolved to form channels with similar specificity.
Structural Studies on Chromatin Remodeling ComplexesIn addition to providing a solution to storing large genomes within the confines of a nucleus, the packaging of eukaryotic DNA into compact chromatin also creates a general mechanism for regulating DNA-associated processes. The tight chromatin organization raises a physical barrier for interactions with factors that mediate transcription, replication, recombination, and repair. Access of these factors to DNA depends on altering the dense packing and positioning of nucleosomes by a number of processes collectively called "chromatin remodeling." This term is used to summarize transitions in chromatin structure that result mainly from two highly interrelated processes: post-translational modification of histone tails and ATP-dependent nucleosome mobilization. We are using both electron microscopy and x-ray crystallography to approach the complex process of chromatin remodeling in a divide-and-conquer fashion. Among the chromatin remodeling complexes we are studying are the SIR and RENT silencing complexes, as well as polycomb repressive complexes.
The monolayer purification technique. A problem with many macromolecular machines, including chromatin remodeling complexes, is their low cellular abundance, making it difficult to isolate sufficient amounts of homogeneous material for structural studies. In vitro reconstitution from recombinant components can prove an even greater barrier. We have been working on an approach, which we named "monolayer purification," to overcome the problem of isolating authentic complexes. The method is based on expression of a His-tagged subunit of the target complex. The cells are then lysed and the extract overlaid with a lipid monolayer spiked with lipids that contain a Ni-NTA headgroup to specifically recruit the complex containing the His-tagged subunit to the monolayer. The monolayer is transferred to a grid and prepared for EM imaging by negative staining or vitrification. By His tagging and adsorbing activators or substrates of a target complex, this method would ensure that only those complexes that are bound to the respective activator or substrate would be imaged, potentially reducing sample heterogeneity and simplifying structure determination by cryo-EM. To extend this approach, we are using His-tagged calmodulin as an adapter to recruit TAP-tagged proteins to Ni-NTA lipid-containing monolayers. This extension will make libraries of TAP-tagged proteins, in addition to libraries of His-tagged proteins, immediately amenable to structure determination by cryo-EM and may pave the way to high-throughput EM studies of macromolecular complexes.
This work is also supported by grants from the National Institutes of Health.
Last updated November 30, 2009
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