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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Membrane proteins are sequestered in a hydrophobic lipid environment, and are therefore resistant to characterization by traditional biochemical techniques which occur in aqueous solution. Traditionally, detergents have been used to solubilize membrane proteins, but these surfactants have detrimental effects on protein form and function. In this thesis, the form, function, and potential mechanism of membrane protein complexes are investigated in detergent-free buffer. Our first study is motivated by the lack of flexible reconstitution scaffolds that maintain a lipid-protein environment for membrane protein stabilization. To this end, we design the peptidisc, a simple method for the universal stabilization of membrane protein complexes in detergent-free solution. Analysis of 5 different membrane protein complexes reconstituted in peptidiscs demonstrate that the method maintains function and increases stability of incorporated membrane proteins. We extend the method to reconstitute the entire membrane proteome of the bacterium Escherichia coli, demonstrating the peptidisc as a “one-size fits all” scaffold. We measure the co-fractionation of reconstituted proteins to identify proteins complexes captured in the peptidisc. The method provides a high-throughput, detergent free approach for identifying the protein-protein interactions of a biological membrane. In the final study, we discover that conformational flexibility of the maltose importer MalFGK2 chloride channel activity in the maltose importer MalFGK2 is linked directly to the transient chloride channel activity. This finding provides support for the expanded alternating access model, which include discrete substeps in the transport model. I discuss the ramifications of the expanded alternating access model on membrane transporter and channel evolution and function. In addition, the development of the peptidisc method is discussed in the broader context of current, detergent free methods for analysis of membrane protein structure, function and interactions.
The outer membrane of Gram-negative bacteria acts as a physical barrier against the dangers of the extracellular environment. The outer membrane contains a number of porins and transporters to facilitate the import of nutrients while simultaneously protecting cells from extracellular assault. How these proteins transport nutrients and how they can be subverted are still areas of investigation. In the first study the mechanisms of transport through the vitamin B₁₂ transporter BtuB is investigated. BtuB was found to interact in a 1:1 molar ratio with the inner membrane protein TonB, which is required for transport of vitamin B₁₂ (cobalamin). Binding of TonB, in turn, alters the binding dynamics of the ligand with BtuB and slows the dissociation of ligand. In the second study transport of the antimicrobial protein colicin E3 across the outer membrane was investigated. Colicin E3 is a ribosomal nuclease that exists in a complex with an inhibitor, immunity protein Im3. Denaturation of colicin E3 was found to facilitate the interaction of the colicin with its outer membrane binding partners by dissociation of Im3. Release of Im3 from colicin E3 allows the nuclease domain of colicin E3 to interact with lipopolysaccharide as part of the transport process. Finally, OmpC and HslT from the Gram-negative Salmonella enterica serovar Typhimurium are hypothesized to interact to protect persistent infectious cells from the oxidizing assault of the immune system. No direct interaction between OmpC and HslT was detectable, possible explanations for this lack of interaction are discussed. These results are discussed in the context of how both ligands and antimicrobial compounds are transported across the outer membrane.
ATP-binding cassette (ABC) transporters couple ATP hydrolysis to import and export of a large array of substances across cell membranes in all kingdoms of life. Since the transport reaction consumes cellular energy, substrate translocation mediated by ABC transporters must be regulated according to the requirements of the cell. This thesis uses the Escherichia coli maltose transporter MalFGK2 to understand the regulatory mechanisms of ABC importers. Biochemical and biophysical approaches were employed to investigate how this transport process is modulated by maltose, the maltose-binding protein MalE and the glucose-specific enzyme EIIAGlc. First, I show that ATP facilitates MalE binding to MalFGK2, which forms the complex of MalE-MalFGK2 for efficient maltose transport. In addition, when the external maltose level exceeds that required, maltose is able to limit the maximal transport rate by promoting dissociation of MalE from MalFGK2. Finally, I find that the N-terminal tail of EIIAGlc and acidic phospholipids are essential for the binding of the protein to the MalK dimer, so that cleavage of ATP by MalFGK2 is inhibited. These results, combined with previous genetic, biochemical and structural work, provide valuable insights into our understanding of the regulatory mechanisms of the maltose transport system.
The SecY protein-conducting channel associates with different cytosolic partners to drive the translocation of preprotein substrates across the bacterial inner membrane. In this thesis, several outstanding questions regarding the structure and function of the SecY channel are addressed. Our first study is motivated by the poorly defined interactions between the channel and its binding partners. We characterize the binding mode and stoichiometry of two SecY interacting proteins, the SecA ATPase and Syd, which each form 1:1 complexes with the channel. In the second study, we isolate the SecY dimer (i.e. two SecY channels), which is shown to be essential to activate the SecA ATPase activity and support protein transport. Analysis of SecY dimers in vivo further demonstrates that each constituent SecY copy has a different role in the translocation reaction. Finally, we discover that the SecY channel, in addition to transporting preprotein substrates, is also highly specific for monovalent anions. This selective conductance explains why translocation does cause a general membrane permeability and cell death. Our findings are discussed in the broader context of genetic, biochemical and structural information on the SecY channel and other translocation components.
Master's Student Supervision (2010 - 2018)
Membrane proteins play significant roles in fundamental biological processes, such as transport of molecules across the membrane, triggering intracellular signaling, maintenance of cell structure and utilization of energies. They are known to comprise about 30 % of genes of entire genome; they constitute around 60 % of current drug targets. Despite their importance, our knowledge about membrane proteins lags far behind that about the soluble proteins. This is chiefly because of their nature that hydrophobic domains of integral membrane proteins are embedded within the phospholipid bilayer and because of the fact that they are generally unstable following extraction from their native membrane environment by detergents. It is also true that available techniques for purifying, analyzing and handling membrane proteins are optimized for water-soluble proteins. Hence, a strategy to solubilize membrane proteins in vivo in structurally relevant conformations by fusing membrane proteins with membrane scaffold protein (MSP) could be an alternative to detergent extraction and in vitro solubilization, allowing the direct expression of soluble membrane proteins in living cells. The MSP, which has an amphipathic helical domain, is thought to protect or shield the hydrophobic transmembrane domain of membrane proteins by sequestering them from aqueous environment. To explore this strategy, an ATP binding cassette (ABC) transporter, MsbA, a known lipid flippase was initially chosen as a model protein. MsbA was fused to maltose binding protein (MBP) and MSP at its N-terminus to increase its expression level and to promote solubilization respectively. It was shown that MsbA fusion construct (MBP-MSP-MsbA) was produced in an appreciable amount in water soluble form post detergent wash in the oligomerization state of a functional dimer and was able to hydrolyze ATP.
TonB-dependent transporters are β-barrel outer membrane proteins that depend on interactions with the inner membrane protein TonB to drive import of scarce nutrients. Upon becoming ligand-loaded, TonB-dependent transporters bind TonB through a β-strand exchange. FhuA is the TonB-dependent transporter that transports hydroxamate iron siderophores, such as ferrichrome and ferricrocin, into the periplasm and also acts as the receptor and transporter of the antimicrobial protein colicin M. The interactions of FhuA with TonB have previously been investigated in detergents, which can affect the conformations of TonB-dependent transporters and alter their interaction with TonB. To exclude the potential negative effects of detergent, FhuA was reconstituted into Nanodiscs that reconstitute a membrane-like environment suitable for biochemical analysis. Binding of TonB to FhuA was found to be strongly dependent on the ligand-loaded state of FhuA. The binding affinity was relatively high (~200 nM) and enthalpy driven, suggesting a disorder-to-order interaction occurring during the β-strand exchange. Colicin M also bound Nanodisc reconstituted FhuA with a high affinity (~3.5 nM) through an entropy driven interaction that may reflect a hydrophobic interaction. The ligand ferricrocin inhibited the binding of colicin M to FhuA. While TonB is required for transport of colicin M into cells, colicin M was not observed to cause the recruitment of TonB to FhuA. Finally, the conformation of FhuA was investigated in the presence of ferricrocin and colicin M by partial proteolysis.
The insoluble nature of membrane proteins has complicated the identification of their interactomes. The Nanodisc has allowed the membrane and membrane proteins to exist in a soluble state. In this thesis, we combined Nanodisc and proteomics and applied the technique to discover the interactome of membrane proteins. Using the SecYEG and MalFGK membrane complex incorporated into Nanodisc, we identified, Syd, SecA, and MalE. These interactions were identified with high specificity and confidence from total soluble protein extracts. The protein YidC was also tested but no interactors were detected. Overall, these results showed that the technique can identify periplasmic and cytosolic interacting partners with high degree of specificity. In a second approach, the method was applied to detect proteins with high affinity for lipid using S. cerevisiae as a model organism. Using Nanodiscs containing different types of phospholipids, many known lipid interactors were identified, including: Ypt1, Sec4, Vps21, Osh6, and Faa1. Interestingly, Caj1 was identified as a PA specific interactor and this interaction was found to be pH dependent. Liposome sedimentation assay showed that Caj1 has affinity for acidic phospholipids. In vivo analysis confirmed the plasma membrane localization of N’-GFP-Caj1 and specifically to the yeast buds. However, pH dependent localization was not observed. Together, with the in vivo and in vitro results suggests that Caj1 is an acidic phospholipid interacting protein.