{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/120335"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/120335","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Investigating strategies to combat gram-negative bacterial antibiotic resistance using molecular simulation","abstract":"Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-05-01","abstract_html":"Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-05-01","abstract_has_math":false,"creators":["Vasan, Archit"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Quant Biology","degree_department":null,"school":null,"contributors":["Tajkhorshid, Emad","Pogorelov, Taras","Shukla, Diwakar","Hergenrother, Paul J"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-05","date_published":"2023-05","updated_at":"2026-07-22T22:24:57Z","subjects":["Antibiotic Resistance","Molecular Simulation"],"languages":["en","eng"],"rights":["Copyright 2023 Archit Vasan"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/120335","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Tajkhorshid, Emad","Pogorelov, Taras","Shukla, Diwakar","Hergenrother, Paul J"]},{"key":"dc:creator","label":"Author","values":["Vasan, Archit"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-05","2023-01-30"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Quant Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Antibiotic Resistance","Molecular Simulation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2023 Archit Vasan"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/120335"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-05-01","The student, Archit Vasan, accepted the attached license on 2023-01-27 at 00:48.","The student, Archit Vasan, submitted this Dissertation for approval on 2023-01-27 at 00:58.","This Dissertation was approved for publication on 2023-01-30 at 10:44.","DSpace SAF Submission Ingestion Package generated from Vireo submission #18831 on 2023-09-01 at 17:12:43","Gram-negative bacteria pose a significant public health concern since they are increasingly becoming resistant to currently available antibiotics, and the development of new antibiotics targeting these bacteria has been very slow. A major difficulty is designing antibiotics that can readily permeate the dense outer membrane (OM) of these bacteria that is composed of various glycolipids known as lipopolysaccharides (LPS). Due to the impermeability of the OM, most essential nutrients for the bacteria diffuse into the cell through a variety of general diffusion, β-barrel OM porins, which are also typically the main entrances for antibiotics into Gram-negative bacteria. Thus, understanding the permeation properties of OM porins would be instrumental to understanding how to develop new antibiotics. The permeability of many OM porins such as OmpF has been hypothesized to be influenced by gating processes; however, a thorough understanding of the underlying mechanisms for these processes is still lacking. This gating behavior has been suggested to be mediated by the dynamics of an internal loop, L3; however, support for this hypothesis remains limited. Using extensive molecular dynamics simulations and an advanced analysis technique known as Markov state models (MSM), we obtain the conformational landscape of L3 that describe the thermodynamic and kinetic information of the complete gating process. MSMs are a class of model used to describe the long timescale dynamics of molecular systems and to obtain the thermodynamic and kinetic information about dynamic processes from the molecular dynamics (MD) simulation data. We find that the gating transitions occur by a large-scale motion of L3, mediated by its two acidic residues, E117 and D121. Movement of E117 to a cluster of basic residues (B-face) in the wall of the porin initiates the transition from the open state, while the movement of D121 to the B-face mediates complete closure of the pore. Additionally, mutations of several B-face residues to neutral or acidic side chains have been shown experimentally to increase substrate permeability. MD simulations performed on these mutants suggest that they reduce the E117 and D121 attraction towards the B-face and hence decrease the probability of pore closure and an increased likelihood of the open state. This hypothesis was further tested by introducing mutations in the B-face residues of OmpF. Furthermore, the key residues involved in the gating transitions are highly conserved across OM porins from various Gram-negative bacteria suggesting the generality of our results. Overall, we propose a thermodynamic and kinetic model as to how the permeability of OM porins depends on a dynamic equilibrium between open and closed conformations. The model provides novel insight on the mechanisms by which OM porins might confer antibiotic resistance to Gram-negative bacteria. Additionally, these results provided a stable open conformation of the porin to use for additional studies such as antibiotic permeation. Using the open state obtained from our studies on gating, we sought to determine mechanisms of antibiotic permeation. Although molecular dynamics (MD) simulations can provide key structural information on molecular processes, using equilibrium or enhanced sampling MD simulations to adequately sample the multiple slow degrees of freedoms (DOFs) in antibiotic permeation processes is computationally intractable. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo-based algorithm to probe more efficiently the permeation pathways of antibiotics through OmpF. The resulting pathways are then used for free energy calculations. The ABC transporter LolCDE is responsible for the transport of lipoprotein across the inner membrane. Furthermore, it has been demonstrated that loss of transport of lipoproteins to the OM can result in cell death. In this context, a prime target for new antibiotics is LolCDE, an ATP-binding cassette (ABC) transporter found exclusively in the inner membrane of Gram-negative bacteria, which transports triacylated lipoproteins to the OM. We use a combination of molecular docking and simulation to understand mechanisms of inhibitor binding and inhibition mechanisms for a novel inhibitor."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Investigating strategies to combat gram-negative bacterial antibiotic resistance using molecular simulation"]}]}],"canonical_facts":{"dc:contributor":["Tajkhorshid, Emad","Pogorelov, Taras","Shukla, Diwakar","Hergenrother, Paul J"],"dc:creator":["Vasan, Archit"],"dc:date":["2023-05","2023-01-30"],"dc:description":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-05-01","The student, Archit Vasan, accepted the attached license on 2023-01-27 at 00:48.","The student, Archit Vasan, submitted this Dissertation for approval on 2023-01-27 at 00:58.","This Dissertation was approved for publication on 2023-01-30 at 10:44.","DSpace SAF Submission Ingestion Package generated from Vireo submission #18831 on 2023-09-01 at 17:12:43","Gram-negative bacteria pose a significant public health concern since they are increasingly becoming resistant to currently available antibiotics, and the development of new antibiotics targeting these bacteria has been very slow. A major difficulty is designing antibiotics that can readily permeate the dense outer membrane (OM) of these bacteria that is composed of various glycolipids known as lipopolysaccharides (LPS). Due to the impermeability of the OM, most essential nutrients for the bacteria diffuse into the cell through a variety of general diffusion, β-barrel OM porins, which are also typically the main entrances for antibiotics into Gram-negative bacteria. Thus, understanding the permeation properties of OM porins would be instrumental to understanding how to develop new antibiotics. The permeability of many OM porins such as OmpF has been hypothesized to be influenced by gating processes; however, a thorough understanding of the underlying mechanisms for these processes is still lacking. This gating behavior has been suggested to be mediated by the dynamics of an internal loop, L3; however, support for this hypothesis remains limited. Using extensive molecular dynamics simulations and an advanced analysis technique known as Markov state models (MSM), we obtain the conformational landscape of L3 that describe the thermodynamic and kinetic information of the complete gating process. MSMs are a class of model used to describe the long timescale dynamics of molecular systems and to obtain the thermodynamic and kinetic information about dynamic processes from the molecular dynamics (MD) simulation data. We find that the gating transitions occur by a large-scale motion of L3, mediated by its two acidic residues, E117 and D121. Movement of E117 to a cluster of basic residues (B-face) in the wall of the porin initiates the transition from the open state, while the movement of D121 to the B-face mediates complete closure of the pore. Additionally, mutations of several B-face residues to neutral or acidic side chains have been shown experimentally to increase substrate permeability. MD simulations performed on these mutants suggest that they reduce the E117 and D121 attraction towards the B-face and hence decrease the probability of pore closure and an increased likelihood of the open state. This hypothesis was further tested by introducing mutations in the B-face residues of OmpF. Furthermore, the key residues involved in the gating transitions are highly conserved across OM porins from various Gram-negative bacteria suggesting the generality of our results. Overall, we propose a thermodynamic and kinetic model as to how the permeability of OM porins depends on a dynamic equilibrium between open and closed conformations. The model provides novel insight on the mechanisms by which OM porins might confer antibiotic resistance to Gram-negative bacteria. Additionally, these results provided a stable open conformation of the porin to use for additional studies such as antibiotic permeation. Using the open state obtained from our studies on gating, we sought to determine mechanisms of antibiotic permeation. Although molecular dynamics (MD) simulations can provide key structural information on molecular processes, using equilibrium or enhanced sampling MD simulations to adequately sample the multiple slow degrees of freedoms (DOFs) in antibiotic permeation processes is computationally intractable. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo-based algorithm to probe more efficiently the permeation pathways of antibiotics through OmpF. The resulting pathways are then used for free energy calculations. The ABC transporter LolCDE is responsible for the transport of lipoprotein across the inner membrane. Furthermore, it has been demonstrated that loss of transport of lipoproteins to the OM can result in cell death. In this context, a prime target for new antibiotics is LolCDE, an ATP-binding cassette (ABC) transporter found exclusively in the inner membrane of Gram-negative bacteria, which transports triacylated lipoproteins to the OM. We use a combination of molecular docking and simulation to understand mechanisms of inhibitor binding and inhibition mechanisms for a novel inhibitor."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/120335"],"dc:language":["en","eng"],"dc:rights":["Copyright 2023 Archit Vasan"],"dc:subject":["Antibiotic Resistance","Molecular Simulation"],"dc:title":["Investigating strategies to combat gram-negative bacterial antibiotic resistance using molecular simulation"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Biophysics & Quant Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:57Z"}