Multidrug efflux pumps are membrane proteins that actively transport foreign objects out of cells. The active efflux of these pumps is a critical self-defense mechanism that enables the survival of bacteria under hostile environments. Efflux pump AcrB is a member of the Resistance-Nodulation-Division (RND) super family. In E. coli, it associates with periplasmic protein AcrA and outer membrane channel TolC to extrude a variety of noxious compounds out of cell from both the cytoplasm and the periplasm. My dissertation research focused on two aspects of this multidrug efflux pump: the oligomerization process during the biogenesis of AcrB and its degradation. Oligomerization is an important aspect of the structure and function for many proteins and has been the subject of many studies. However, most of such studies focused on soluble proteins. The oligomerization process of membrane proteins, including AcrB, is rarely explored. In chapter 2, the co-assembly of AcrB variants co-expressed in the same cell was used as a tool to investigate the assembly of AcrB trimers during its bio-production. By monitoring the portion of pure trimers containing only one type of subunit and hybrid trimers containing a mixture of the two kinds of subunits, it was found that the oligomerization of membrane proteins is not a random process as the formation of pure trimer is favored. In chapter 3, the GALLEX system was used to monitor AcrB oligomerization in cells under the native condition. Previously GALLEX has only been used to monitor the oligomerization of small transmembrane proteins. By constructing a series of fusion proteins with different linker length between LexA and AcrB, and optimizing inducer concentration, we finally developed a system that could be used to differentiate AcrB trimers of different oligomerization affinities. While chapters 2 and 3 focus on the trimerization of AcrB, a critical step of its biogenesis, chapters 4 and 5 focus on its life time and degradation. In chapter 4, the life time of AcrB was measured by incorporating non-natural amino acid azidohomoalanine (AHA) into protein translation. Using this method, it was determined that that the half-life of both AcrA and AcrB in E. coli were six days. The surprisingly long lifetime of these detoxification proteins might represent a strategy by the bacteria to conserve energy and maximize their competition niche for survival in a hostile environment. In chapter 5, the degradation process of ssra tagged AcrB was investigated. In-vivo degradation test showed that properly inserted AcrB can be digested after addition of ssra-tag to its C-terminus. It was found that cytoplasmic unfoldase-protease complex ClpXP and chaperone SspB are involved in the degradation. In vitro assay is still being optimized to quantitatively analyze the activity of ClpXP in the degradation of AcrB.

Multiple drug resistance (MDR) has been a severe issue in treatment and recovery from infection.Gram-negative bacteria intrinsically exhibit higher drug tolerance than Gram-positive microbes. In this thesis, two proteins involved in Gram-negative bacterial MDR were studied, AcrB and SurA. Resistance-nodulation-cell division pump AcrAB-TolC is the major MDR efflux system in Gram-negative bacteria and efficiently extrudes a broad range of substances from the cells. To study subtle conformational changes of AcrB in vivo, a reporter platform was designed. Cysteine pairs were introduced into different regions in the periplasmic domain of the protein, and the extents of disulfide bond formation were examined. Using this platform, an inactive mutant, AcrB∆loop, was created that existed as a well-folded monomer in vivo. Next, random mutageneses were performed on a functionally compromised mutant, AcrBP223G, to identify residues that restored the function loss. The mechanism of function restoration was examined. SurA is a periplasmic molecular chaperone for outer membrane biogenesis. Deletion of SurA decreased outer membrane density and bacterial drug resistance. The dependence of SurA function on structural flexibility and stability was examined. In addition, the effect of molecular crowding on SurA interaction with its outer membrane protein substrates was examined.

Drug resistant bacteria pathogen poses a severe threat to human health. Bacterial drug efflux pumps are transporter proteins involved in the export of antibiotics out of cells. Efflux by transporters is one of the major drug resistant mechanisms. Multidrug efflux pumps can transport multiple classes of antibiotics and are associated with bacteria multiple drug resistance (MDR). Overproduction of these pumps reduces susceptibility of bacteria to a variety of antibiotics. MDR regulators are cytoplasmic proteins that control the expression level of MDR transporters in response to the cellular concentration of antibiotics. This thesis research focuses on three main directions in the area of bacteria drug resistance: the structural and functional study of a MDR transporter, the characterization of a novel MDR regulator protein, and the development of a sensing method for the detection of glycopeptide antibiotics. Acriflavine resistance protein B (AcrB) in Escherichia coli belongs to resistance nodulation division (RND) superfamily of efflux transporters. It plays an important role in confering multidrug resistance in Gram-negative bacteria. The functional unit of AcrB is a trimer in vivo. However, the relationship between AcrB trimer stability and functionality remains elusive. In chapter 2, a residue that is critical for AcrB trimerization, Pro 223, was identified. The replacement of Pro 223 by other residues destabilized AcrB trimer, and thus decreased its activity. The loss of transport activity could be partially recovered when the AcrB trimer was stabilized by the introduction of a pair of inter-subunit disulfide bond. In chapter 3, a systematically alanine-scanning study of the producing loop (amino acid residues 211-240) was conducted. Five residues in the loop were found to be important for AcrB activity. These residues form a collar or belt in the loop close to the tip. These mutation studies revealed new insight into the conformation of the loop during AcrB trimerization. In chapter 4, residue Arg 780 was identified to be crucial for the pump function of AcrB. The study results indicated that Pro 223 serves as a “wedge” and Arg 780 as a “lock” via hydrogen bonding between the backbone carbonyl oxygen of Pro 223 and side chain of Arg780. Similar as Pro 223, replacement of Arg 780 by other residues drastically decreased the activity of AcrB. Dissociation of the AcrB trimer also contributed to the decrease of activity. However, the introduction of inter-subunit disulfide bond could not restore the function of the mutant, indicating that Arg 780 plays multiples roles in the operation of AcrB. In chapter 5, a MDR regulator ST1710 from the archaeon Sulfolobus tokodaii, homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators, was characterized in vitro. The binding affinities of ligands and double strand (ds) DNA for ST1710 were measured. The presence of substrates suppressed the interaction between ST1710 and dsDNA, which indicated that ST1710 functioned as a repressor in vivo. Finally, in chapter 6, a direct fluorescence polarization based method for the detection of glycopeptide antibiotics is developed. Briefly, the acetylated tripeptide L-Lys-D-Ala-D-Ala was labeled with a fluorophore (fluorescein isothiocyanate or AlexaFluor 680) to create a peptide probe. The fluorescence polarization signal of the peptide probe increased upon binding with glycopeptide antibiotics in a concentration dependent manner. The detection is highly selective toward glycopeptide antibiotics. The designed method is expected it to have broad applications in both research and clinical settings.

The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms. The first direction of my research is in the area of the biogenesis of the bacterial outer membrane. The outer membrane serves as a permeability barrier in Gram-negative bacteria. Antibiotics cross the membrane barrier mainly via diffusion into the lipid bilayer or channels formed by outer membrane proteins. Therefore, bacterial drug resistance is closely correlated with the integrity of the outer membrane, which depends on the correct folding of the outer membrane proteins. The folding of the outer membrane proteins has been studied extensively in dilute buffer solution. However, the cell periplasm, where the folding actually occurs, is a crowded environment. In Chapter 2, effects of the macromolecular crowding on the folding mechanisms of two bacterial outer membrane proteins (OmpA and OmpT) were examined. Our results suggested that the periplasmic domain of OmpA improved the efficiency of the OmpA maturation under the crowding condition, while refolding of OmpT was barely affected by the crowding. The second direction of my research focuses on the major multidrug efflux transporter in Gram-negative bacteria, AcrB. AcrB is an obligate trimer, which exists and functions exclusively in a trimeric state. In Chapter 3, the unfolding of the AcrB trimer was investigated. Our results revealed that sodium dodecyl sulfate induced unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild type AcrB. In Chapter 4, the correlation between the AcrB trimer stability and the transporter activity was studied. A non-linear correlation was observed, in which the threshold trimer stability was required to maintain the efflux activity. Finally, in Chapter 5, the stability of another inner membrane protein, AqpZ, was studied. AqpZ was remarkably stable. Several molecular engineering approaches were tested to improve the thermal stability of the protein.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants. Due to their properties, PCBs accumulate in the food-chain and post a threat to the health of human beings and wildlife. Hydroxylated PCBs (OH-PCBs) are oxidative metabolites of PCBs and are more hydrophilic than their parent PCBs. One of the best approaches to break down these contaminants is through bioremediation, which is an environmental friendly process that uses microorganisms to restore natural environment. Towards this goal, we have investigated the toxicity and accumulation of PCBs and OH-PCBs in a Gram-negative bacterium, Escherichia coli. We have also determined the role played by a primary multidrug efflux transporter AcrB on the accumulation of PCBs and OH-PCBs in bacterial cell. We found that one of the PCBs tested was toxic to E. coli, while different OH-PCBs have different levels of toxicity; the acrB knockout strain accumulated significantly more PCBs and OH-PCBs than the wild-type strain, suggesting that these compounds are substrates of the efflux pump; higher cytoplasmic concentrations of OH-PCBs were also observed in the acrB knockout strain using the biosensors. Based on these observations, we conclude that both PCBs and OH-PCBs are substrates of protein AcrB. Therefore the efflux activities of multidrug resistant pumps in Gram-negative bacteria should be considered while designing bioremediation approaches.

abstract: Emergence of multidrug resistant (MDR) bacteria is a major concern to global health. One of the major MDR mechanisms bacteria employ is efflux pumps for the expulsion of drugs from the cell. In Escherichia coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, a trimeric membrane protein is well-known for its substrate promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. Newly identified AcrB residues were shown to be functionally relevant in the drug binding and translocation pathway using a positive genetic selection strategy. These residues—Y49, V127, D153, G288, F453, and L486—were identified as the sites of suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. Using site-directed mutagenesis (SDM) along with the real-time efflux and the classical minimum inhibitory concentration (MIC) assays, I was able to characterize the mechanism of suppression. Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression. To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A. / Dissertation/Thesis / Masters Thesis Biology 2016

Escherichia coli transporter protein AcrB and its homologues are the inner membrane components of the Resistance-Nodulation-Division (RND) family efflux pumps in Gram-negative bacteria. It is well accepted that soluble proteins are only marginally stable, but such insight is missing for membrane proteins. The lack of stability data, including thermodynamic stability and oligomer association affinity is a result of intrinsic difficulties in working with membrane proteins. In addition, the degradation of soluble proteins in E. coli has been extensively studied whereas the degradation process of membrane proteins remains unclear. A focus of my thesis is the validation and development of methods used to measure the thermo- and oligomeric- stability of membrane proteins. I investigated the mechanism of a popular thermal-stability assay developed specifically for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). I found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal stability assay. The observed fluorescence increase is likely caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding. I then applied these methods in the study of three projects. In the first project, I investigated how suppressor mutations restore the function of AcrBP223G, in which the Pro223 to Gly mutation compromised the function of AcrB via disrupting AcrB trimerization. The results suggested that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding. In the second project, I created two AcrB fusion proteins, with C-terminal yellow fluorescence protein (YFP) and cyan fluorescence protein (CFP), respectively. YFP and CFP form a fluorescence resonance energy transfer (FRET) pair. Using this pair of fusion proteins, I studied AcrB assembly both in detergent micelles and in lipid bilayers. A positive cooperativity was observed in kinetic studies of association of AcrB trimer. Reconstitution experiment revealed that the association showed a higher FRET efficiency and faster association rate in liposome than in DDM. In the last project, I developed a fluorescence method to study the degradation of AcrB-ssrA by the ClpXP system. Comparing to the degradation of GFP-ssrA, degradation of AcrB-CFP-ssrA showed a lower maximum velocity and tighter binding to the enzymes with a positive cooperativity.

This work was aimed at developing novel tools that utilize HINT, an empirical forcefield capable of quantitating both hydrophobic and hydrophilic (hydropathic) interactions, for implementation in theoretical biology and drug discovery/design. The role of hydrophobicity in determination of macromolecular structure and formation of complexes in biological molecules is undeniable and has been the subject of research across several decades. Hydrophobicity is introduced, with a review of its history and contemporary theories. This is followed by a description of various methods that quantify this all-pervading phenomenon and their use in protein folding and contemporary drug design projects – including a detailed overview of the HINT forcefield. The specific aim of this dissertation is to introduce our attempts at developing new methods for use in the study of antibacterial drug resistance and antiviral drug discovery. Multidrug efflux is commonly regarded as a fast growing problem in the field of medicine. Several species of microbes are known to have developed resistance against almost all classes of antibiotics by various modes-of-action, which include multidrug transporters (a.k.a. efflux pumps). These proteins are present in both gram-positive and gram-negative bacteria and extrude molecules of various classes. They protect the efflux pump-expressing bacterium from harmful effects of exogenous agents by simply evacuating the latter. Perhaps the best characterized mechanism amongst these is that of the AcrA-AcrB-TolC efflux pump. Data is available in literature and perhaps also in proprietary databases available with pharmaceutical companies, characterizing this pump in terms of the minimum inhibitory concentration ratios (MIC ratios) for various antibiotics. We procured a curated dataset of 32 β-lactam and 12 antibiotics of other classes from this literature. Initial attempts at studying the MIC ratios of β-lactam antibiotics as a function of their three dimensional topology via 3D-quantitative structure activity relationship (3D-QSAR) technology yielded seemingly good models. However, this methodology is essentially designed to address single receptor-ligand interactions. Molecules being transported by the efflux pump must undoubtedly be involved in multiple interactions with the same. Notably, such methods require a pharmacophoric overlap of ligands prior to the generation of models, thereby limiting their applicability to a set of structurally-related compounds. Thus, we designed a novel method that takes various interactions between antibiotic agents and the AcrA-AcrB-TolC pump into account in conjunction with certain properties of the drugs. This method yielded mathematical models that are capable of predicting high/low efflux with significant efficiency (>93% correct). The development of this method, along with the results from its validation, is presented herein. A parallel aim being pursued by us is to discover inhibitors for hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 3 (HPIV3) by in silico screening. The basis for targeting HN is explored, along with commentary on the methodology adopted during this effort. This project yielded a moderate success rate of 34%, perhaps due to problems in the computational methodology utilized. We highlight one particular problem – that of emulating target flexibility – and explore new avenues for overcoming this obstacle in the long run. As a starting point towards enhancing the tools available to us for virtual screening in general (and for discovering antiviral compounds in specific), we explored the compatibility between sidechain rotamer libraries and the HINT scoring function. A new algorithm was designed to optimize amino acid residue sidechains, if provided with the backbone coordinates, by generating sidechain positions using the Dunbrack and Cohen backbone-dependent rotamer library and scoring them with the HINT scoring function. This rotamer library was previously used by its developers previously to design a very successful sidechain optimization algorithm called SCWRL. Output structures from our algorithm were compared with those from SCWRL and showed extraordinary similarities as well as significant differences, which are discussed herein. This successful implementation of HINT in our sidechain optimization algorithm establishes the compatibility between this forcefield and sidechain rotamer libraries. Future aims in this project include enhancement of our current algorithm and the design of a new algorithm to explore partial induced-fit in targets aimed at improving current docking methodology. This work shows significant progress towards the implementation of our hydropathic force field in theoretical modeling of biological systems in order to enhance our ability to understand atomistic details of inter- and intramolecular interactions which must form the basis for a wide variety of biological phenomena. Such efforts are key to not only to understanding the said phenomena, but also towards a solid basis for efficient drug design in the future.