Microscopic characterization of beta-barrel proteins using atomic scale simulations

Haloi, Nandan

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https://hdl.handle.net/2142/113822 Copy

Description

Title

Microscopic characterization of beta-barrel proteins using atomic scale simulations

Author(s)

Haloi, Nandan

Issue Date

2021-10-18

Director of Research (if dissertation) or Advisor (if thesis)

Tajkhorshid, Emad

Doctoral Committee Chair(s)

Tajkhorshid, Emad

Committee Member(s)

• Kwok, Wai-meng
• Hergenrother, Paul
• Pogorelov, Taras

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Quant Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Molecular dynamics simulations, beta barrels, outer-membrane, free-energy calculations, modeling

Abstract

Beta-barrel proteins are found in the outer membrane of bacteria, mitochondria and chloroplast. These proteins serve essential functions including acting as porins, transporters, enzymes, and receptors. This dissertation focuses on using and developing computational methods to investigate functional aspects of two -barrel proteins that serve as porins: (i) bacterial outer membrane (OM) protein, OmpF, and (ii) mitochondrial OM channel, VDAC. Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their OM. Antibiotics crossing the OM typically do so through OM porins, such as OmpF, the most abundant porin of Escherichia coli; therefore, understanding mechanisms facilitating this permeation would aid in antibiotic development. Primary amine-appended antibiotics are known to be highly permeable through this porin; however, there is a lack of understanding of the molecular basis of this phenomenon. Therefore, I used molecular dynamics (MD) to compare the free energy of permeation of antibiotics with and without primary amine through OmpF. This project was challenging, because antibiotic permeation processes involve multiple slow degrees of freedoms (DOFs) of the antibiotic that are intractable to sample in typical enhanced sampling calculations. To overcome this challenge, I developed a novel, computationally efficient algorithm, Monte Carlo based Pathway Search (MCPS), to focus sampling on all the relevant slow DOFs and then use this exhaustive sampling to determine an optimal permeation pathway. This path was then used for one-dimensional bias exchange umbrella sampling simulations. The free-energy barrier for permeation was found to be significantly lower for aminated drugs corresponding to a greater permeability through OM porins. Further analysis revealed that the amine facilitates favorable antibiotic permeation through porins by enabling the antibiotic to align its dipole to the intrinsic electric field of the porin and then hop along the pore to form interactions with several charged residues. The importance of these interactions in permeation was further validated with mutagenesis and whole-cell accumulation assays. Overall, the insight on the importance of the primary amine o ered by this study could help motivate the design of future antibiotics. The permeability of OM porins has been hypothesized to be influenced by their gating processes; however, a thorough understanding of 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 MD simulations and Markov state analysis, I obtained the conformational landspace of L3 that describes the thermodynamic and kinetic information of the complete gating process. These gating transitions are mediated by movement of acidic residues in L3 to interact with two opposite sides of the barrel wall. Overall, I propose a model for 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. The beta -barrel channel of mitochondria, VDAC, not only acts as the major pathway into and out of mitochondria, but also mediates an intimate dichotomy between metabolism and death by interacting with anti- and pro-apoptotic proteins. For example, VDAC forms complex with cytosolic enzyme hexokinase (HK), a process with plays a crucial role in regulating cell growth and survival; however, structural details of this complex remain elusive. Complex formation involves a priori membrane binding of HK, making structural determination of VDAC/HK complex di cult using typical experimental techniques. Simulation of this process is also challenging, because complex formation occurs on a time-scale not feasibly obtained by conventional MD simulation. Therefore, I designed a hybrid MD and Brownian dynamics (BD) approach: describing first the membrane insertion of HK using MD, and then formation of VDAC/HK complex using BD simulations. The resulting complex substantiates how HK modulates VDAC permeability and how VDAC phosphorylation disrupts HK binding, features that were verified with electrophysiological experiments and have implications in mitochondria-mediated cell death.

Graduation Semester

2021-12

Type of Resource

Thesis

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http://hdl.handle.net/2142/113822

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© 2021 by Nandan Haloi. All rights reserved

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