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Bert van den Berg, Ph.D.Academic Role: Associate Professor
Faculty Appointment(s) In:
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Structure and function of membrane transport proteins Biological membranes form the interface between cells and their environment. Transport of molecules across the membrane barrier is essential for all cells (e.g. for metabolite supply). As a consequence, many (human) diseases, with cystic fibrosis as the best-known example, are caused by mutations in membrane transport proteins. As such they are of enormous medical importance, indicated by the fact that ~60% of pharmaceutical drugs target membrane proteins. Unfortunately, our understanding of how these proteins carry out their tasks is still rather limited. This is mainly due to a lack of detailed structural information. To date only ~200 unique membrane protein structures have been deposited in the Protein Data Bank (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html). This is only ~1% of all known protein structures, despite the fact that membrane proteins comprise ~25% of all open reading frames. This discrepancy is mainly due to the difficulties in obtaining milligram amounts of highly pure membrane proteins, required for structural studies. However, the increase in the number of solved membrane protein structures in recent years demonstrates that structure determination of membrane proteins, although challenging, is definitely feasible. We are interested in determining the 3D structures of bacterial outer membrane proteins by X-ray crystallography. Those structures are then used, in combination with functional data obtained from biochemical experiments, to propose mechanistic models that can be tested experimentally. Please have a look at our structure gallery (Fig. 1). FIGURE 1
The main topics of research in the laboratory are: 1. Transport of hydrophobic molecules across the bacterial outer membrane The outer membrane (OM) of gram-negative bacteria forms as efficient barrier for the permeation of polar and hydrophobic molecules, due to the presence of the lipopolysaccharide (LPS) layer on the outside of the cell. Consequently, many channels are present in the OM for the uptake of water-soluble molecules required for growth and function of the cell. So far, however, members of the FadL protein family are the only channels known that are dedicated to the uptake of hydrophobic molecules, such as long-chain fatty acids (LCFAs) and aromatic hydrocarbons (toluene, benzene, etc.). FadL channel-mediated transport is based on diffusion, and does not require an energized membrane. We have previously determined crystal structures of the E. coli LCFA transporter FadL, and have proposed a transport model based on the presence of detergent molecules in the structures and conformational changes between the structures (1). We have recently expanded our work to aromatic hydrocarbon transporters from biodegrading bacteria, and shown that while these channels are structurally similar to FadL, they do not transport LCFAs (ie FadL channels are substrate-specific; 2). Finally, based on structures of E. coli mutant FadL proteins and in vivo transport assays, we have shown that E. coli FadL transports its substrates according to a novel mechanism, involving lateral diffusion of the substrate through an opening in the wall of the channel, into the outer membrane (3; Fig. 2). FIGURE 2
Fig. 2. Proposed lateral diffision model for the uptake of hydrophobic substrates by FadL channels. a, Substrate (red hexagon) capture from the extracellular medium by a low-affinity binding site (L); b, diffusion of the substrate into an adjacent high-affinity binding site H (blue); c, spontaneous conformational changes in the N terminus (purple) result in substrate release and create a continuous passageway to the barrel wall opening formed by the kink in strand S3. The substrate diffuses laterally through the opening into the outer membrane (OM). The polar part of the LPS, consituting the principal barrier in the transport process, is shown in grey. The extracellular milieu (E) is at the top and the periplasm (P) is at the bottom. Future research (supported by NIH grant 5R01GM074824) on FadL proteins will aim to address the following outstanding questions: 1. Is the transport mechanism as determined for E. coli FadL valid for all FadL channels? 2. What is the substrate specificity of FadL channels? Can we rationalize the substrate specificity in terms of structure? Finally, can be convert a toluene channel into a LCFA transport channel? 3. Which residues are important for substrate binding and release? 4. Are there other channels in the outer membrane that transport hydrophobic compounds (e.g. the OmpW family; 4)? Experimental techniques that are used to address these questions include recombinant DNA technology, membrane protein expression and purification, in vivo transport assays with radiolabeled substrates, in vitro substrate binding assays using fluorescence spectroscopy, and protein structure determination by X-ray crystallography. Because FadL homologues are found in many pathogenic and biodegrading bacteria, FadL channel research may have implications for combating bacterial infections and bioremediating xenobiotics in the environment. 2. Structural and biochemical characterization of the OprD membrane protein family In gram-negative bacteria such as E. coli, the uptake of the majority of small water-soluble compounds is mediated by porins such as OmpF. Many gram-negative bacteria (e.g. pseudomonads), however, do not have porins and consequently have a poorly permeable OM. In these bacteria, transport of most water-soluble compounds is mediated by substrate-specific channels of the OprD family. In the human pathogen Pseudomonas aeruginosa, the OprD family has 19 members that have 45-55% pairwise sequence identity. To gain insight into the substrate transport mechanism of the OprD family members, we have recently determined the first crystal structures of OprD family channels, that of OprD (5; Fig. 1) and OpdK (6), both from Pseudomonas aeruginosa. These structures form the basis for our current and future research regarding this family, with the aim of answering the following questions (supported by NIH grant 1R01GM085785): 1. What is the substrate specificity of OprD channels? To address this issue, in vivo and/or in vitro substrate transport assays will be developed, using an extensive set of radiolabeled substrates. We will also characterize substrate binding by single channel recordings of reconstituted channels in planar lipid bilayers (in collaboration with Liviu Movileanu, Syracuse University; 6). 2. Can we rationalize the substrate specificity in terms of structure? What are the structural determinants of substrate specificity? To answer this question, a number of additional OprD channel structures will be determined by X-ray crystallography, in the absence and presence of substrates. The experimental approaches will be complemented by detailed molecular dynamics simulations and substrate docking studies on OprD channels (in collaboration with Syma Khalid, Southampton University, UK). Answering the above questions will give, for the first time, detailed information about the structural basis of substrate specificity of a group of closely related membrane channels. It is conceivable that such information could be used to generate a "permeability database" in which prospective antibiotics could be screened for their ability to traverse the OM of pathogenic bacteria via OprD channels. In addition to these two major research projects, smaller projects in the lab are focused on autotransporters, OM proteases and two-partner secretion systems. We are also interested in the structures of eukaryotic metal transport proteins.
Office: Biotech 2, Suite 115
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373 Plantation Street Worcester, MA 01605