2004 — 2011 |
Walz, Thomas |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure and Function of Lens Membrane Proteins
DESCRIPTION (provided by applicant): The ocular lens is a unique structure exquisitely designed to focus light onto the retina, a process that requires substantial shape changes of the lens according to the distance of the eye from the object it is focusing on. We are interested in the structure and function of membrane proteins in the lens, which play crucial roles in maintaining lens homeostasis and transparency. Membrane channels and transporters are the basis for a microcirculation system that supplies deeper-lying fiber cells with nutrients and clears them of waste products. Membrane proteins also mediate the tight packing of the fiber cells, thus helping to avoid light scattering by the lens tissue. This proposal focuses on the two major membrane proteins in lens fiber cells, the tetraspanin MP20 and the water channel AQP0. Using two-dimensional (2D) AQP0 crystals, we are also working towards understanding the general principles that underlie non-specific interactions of lipids with membrane proteins. In the previous funding period, we have produced a 1.9 [unreadable] density map of double-layered 2D crystals of AQP0, which resolved the lipid molecules surrounding the protein. Specific Aim 1 of this proposal is to further study non-specific lipid-protein interactions. We will expand the electron crystallographic data set to model alternative conformations of the lipids surrounding AQP0. We will also attempt to extend the resolution of the density map to 1.5 [unreadable], which may allow us to visualize charges of residues in AQP0. We are particularly interested in the charge state of histidines 44 and 60 at different pH values, as these two residues have been implicated in the pH regulation of water conduction by AQP0. In addition, we will visualize AQP0 in 2D crystals grown using lipids with different acyl chains and head groups. These experiments will allow us to systematically investigate the lipid characteristics that define non-specific lipid-protein interactions and to understand how lipids and proteins adapt to each other. We also obtained hexagonal 2D crystals of AQP0, and Specific Aim 2 of this proposal is to use a variety of structural and biophysical methods to elucidate how a protein designed to form tetramers assembling into square arrays can form hexagonal 2D crystals. Structural information on MP20 as well as other tetraspanins is still sparse. Specific Aim 3 is thus to determine the structure of MP20 primarily by electron microscopy but also pursuing X-ray crystallography. The structural information obtained for MP20 may be useful to model the structure of other members of the tetraspanin family, which are important in many cellular processes, such as cell adhesion, proliferation, activation, migration, and apoptosis. In Specific Aim 4 we will characterize the function of MP20 as a cell adhesion molecule by studying its interaction with galectin-3, a prominent adhesion modulator. We will also determine whether MP20 can bind to lens-specific integrins, as many tetraspanins are known to interact with integrins, especially if these contain a ?1 subunit. PUBLIC HEALTH RELEVANCE The importance of aquaporin-0 and MP20 for proper lens function is illustrated by the fact that mutations in either one of these two membrane proteins lead to the formation of cataracts. A wealth of biochemical and biophysical studies has also established the importance of protein-lipid interactions for the assembly, stability, and function of membrane proteins. Almost nothing is known about how lipids affect the structure and function of membrane proteins, whose dysfunction are the cause of a large number of human disorders.
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0.915 |
2007 — 2011 |
Walz, Thomas |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Electron Microscopy Core
The EM core of this Program Project has two related purposes - to support instrumentation essential for the research proposed in the four projects of this grant and to facilitate application of the methods being developed in these projects to collaborative and independent efforts. The principal components of the core are the electron microscopy facilities at Harvard Medical School (HMS) and at Brandeis University. The HMS facility will house four FBI microscopes: a Tecnai F20, a Tecnai T12, a CM10, and a Polara F30. The F30, the F20, and the T12 microscopes are equipped with cryo-stages;the F30 and the F20 have field-emission guns. The Brandeis facility also houses four FBI instruments: a Morgagni M268, and EM20, a CM12, and a Tecnai F30. The last three have cryo-stages;the F30 has a field-emission gun and an energy filter. The core supports the operation of these microscopes and the salary of a facilities manager at each location, as well as necessary ancillary equipment. The core also has a computational component. It maintainsand upgrades all standard EM software, including program suites such as FREALIGN, IMAGIC, and SPIDER, through a structural biology computational grid (SBGrid) established by the Center for Molecular and Cellular Dynamics at HMS. Finally, by providing EM and computational facilities, the core allows the P.I.'s of these projects to collaborate with other groups at various institutionsand to train postdoctoral fellows and students in the relevant methods. Relevance: Electron microscopy is the key experimental tool that bridges between low- and high-resolution structural methods. Large assemblies will be targets of future interventions in human disease, and electron microscopy will be central for relating structure and function of these complexes
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0.915 |
2007 — 2011 |
Walz, Thomas |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Membrane Proteins and Iron Delivery to Cells
Abstract Our long-term objective is to understand the structural basis for the delivery of iron to cells. In particular we will study the structure of the following three proteins: (/) The transferrin (Tf)-transferrin receptor (TfR) complex. We have determined the structure of the Tf-TfR complex using a soluble construct of the TfR ectodomain lacking the stalk region. The resulting structure strongly suggests that the TfR stalk is involved in Tf binding. We will now determine the structure of the complex in the presence of the stalk and perform functional studies to elucidate the effect of the TfR stalk on iron release from the N-terminal lobe of receptor- bound Tf. (//) The divalent metal ion transporter-1 (DMT1). Iron released from the Tf-TfR complex is transported across the endosomal membrane by DMT1, the same protein that mediates iron uptake from the intestinal lumen through the apical surface of duodenal enterocytes. Mutations in DMT1 cause severe hypochromic microcytic anemia and iron overload. We have expressed mg amounts of the DMT1 ortholog from E. co//. We are using this protein to produce two-dimensional (2D) crystals suitable for electron crystallographic structure determination. In parallel, we will attempt to grow three-dimensional (3D) crystals for X-ray crystallographic structure determination and perform structural studies on other bacterial homologs as well as human DMT1. (Hi)Ferroportin. A second iron transporter, ferroportin, exports iron across the basolateral membrane of duodenal enterocytes to the circulation. Mutations in ferroportin cause type IV hemochromatosis, also known as ferroportin disease. We will express human ferroportin for 2D and later for 3D crystallization trials to determine its structure either by electron or X-ray crystallography. We will then decorate ferroportin2D crystals with the peptide hormone hepcidin to elucidate the binding interaction. Relevance Many proteins depend on iron as a co-factor for redox reactions or ligand coordination, making iron an essential element. The facile conversion between ferrous (Fe2+) and ferric iron (Fe3+) poses significant dangers to living cells, however, because it can lead to the formation of hydroxyl radicals, a major source for oxidative damage to proteins, nucleic acids and lipids. Moreover, under physiological conditions ferric iron forms a highly insoluble hydroxide complex, so that despite its abundance, iron is not easily accessible to cells. Toxicity and insolubility have forced the evolution of highly sophisticated machineries for acquiring, storing, and distributing iron. Malfunctioning of these machineries lead either to iron deficiency disorders or iron overload diseases.
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0.915 |
2008 — 2011 |
Walz, Thomas |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural and Functional Studies of Urea Channels
Abstract Urea is the main catabolite in mammals and an important nitrogen source for many microbes. This proposal focuses on structural and functional studies of membrane proteins that facilitate transmembrane urea transport, specifically members of the aquaporin (AQP), urea transporter (UT), and urea/amide channel (UAC) families. We are studying AQP9, which has the broadest substrate specificity among all known AQPs, UreI from Helicobacter pylori, a member of the UAC family, and the urea transporters UT-Apl from Actinobacillus pleuropneumoniae and UT-Ec from the uropathogenic E. coli strain 536. The Specific Aims of this proposal are: (i) to determine the transport kinetics of AQP9 for various solutes. We will perform stopped-flow measurements on AQP9 proteoliposomes to characterize the transport kinetics for various solutes, including water, glycerol and larger solutes. The results will determine the physiological relevance of the AQP9-mediated transport of these solutes. (ii) to solve the structure of AQP9. We have already produced very well ordered two-dimensional (2D) crystals of AQP9 that diffract to about 3.8 [unreadable] resolution. We will continue to pursue electron crystallography of 2D crystals, but also x-ray crystallography of 3D crystals, to produce an atomic model of AQP9. (iii) to determine the transport kinetics of UreI, UT-Apl and UT-Ec for urea and water. We will perform stopped-flow measurements on proteoliposomes containing these urea channels to characterize their transport kinetics. The results will reveal similarities and differences in the function of these proteins. (iv) to obtain structural information on UreI, UT-Apl and UT-Ec. We will use biochemical and electron microscopic techniques to determine the oligomeric state of these urea channels. Our ultimate goal is to produce crystals (2D or 3D) of these proteins that will be suitable for structure determination by electron or x-ray crystallography. Relevance AQP9-mediated glycerol transport out of adipocytes and into the liver may be important to support gluconeogenesis in the fasted state. AQP9 is also permeated by arsenite and might contribute to the toxicity of arsenic ingestion. AQP9 may thus be a target for treating pathophysiological conditions resulting from eating disorders and arsenic poisoning. The availability of a structure for a UT might aid the development of novel diuretic compounds that selectively block urea reabsorption without interfering with the salt balance. UTs also play a crucial role in the survival of human pathogens. An atomic structure of the UT-Apl could thus potentially be used to develop specific inhibitors of bacterial urea transport. Transporters of the UAC family could be particularly potent targets for new antibiotics, since they do not have any homologs in eukaryotes.
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0.915 |
2012 — 2016 |
Walz, Thomas |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 2: Walz
instructions): This component of the Program Project will enhance a new sample preparation method we have developed for single-particle electron microscopy (EM) and apply it to studying the multisubunit tethering complexes (MTCs) that target vesicular carriers of membrane traffic. (1) We have recently developed the monolayer purification and Affinity Grid techniques, which use Ni-NTA lipids in a lipid monolayer to recruit His-tagged target proteins directly from cell extracts. We propose to extend the Affinity Grid repertoire to include capture of proteins with tags other than histidine. In particular, we will test biotinylated lipids to recruit proteins using an avidin adaptor, synthesis of a lipid with a glutathione (D,l-glutamyl-l-cysteinylglycine, GSH) group to recruit proteins with a glutathione-S-transferase (GST) tag, and affinity-tagged Fc fragments to recruit proteins with a tandem affinity purification (TAP) tag. Because of the commercial availability of yeast libraries of TAP-tagged and GST-fusion constructs, the tagged Fc fragments and the GSH-functionalized lipid may allow high-throughput applications of the Affinity Grid. (2) We will continue our structural studies of multisubunit tethering complexes (MTCs), taking advantage of the Affinity Grid approach. MTCs mediate the first contact between a transport vesicle and its target membrane and are thought to orchestrate vesicle capture, docking, and fusion through interactions with Rab GTPases, coat proteins and SNAREs. We have already obtained structures of TRAPPI and II and of the DsH complex and the Cog1-4 subcomplex of COG. By determining the structures of additional MTCs - TRAPPIII, HOPS, GARP, intact COG and exocyst - and analyzing their interactions with Rab GTPases and SNARE proteins, we aim to understand how these MTCs are organized, how their different organizations mediate vesicle tethering, what conformational changes underlie MTC-assisted SNARE complex assembly, and how mutations in subunits interfere with function of MTCs.
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0.915 |