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Investigation of Protein/Ligand Interactions Relating Structural Dynamics to Function: Combined Computational and Experimental Approaches

Pavlovicz, Ryan Elliott
http://rave.ohiolink.edu/etdc/view?acc_num=osu1397220613

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Biophysics.
The use of computers in chemistry has matured significantly since the introduction of the modern personal computer, leading to the development of many tools that may be used to describe phenomena that are difficult or otherwise impossible to observe experimentally. Computational chemistry is becoming an integral component of many research programs, often resulting in the formation of hypotheses that may be tested experimentally. This document details the application of computational tools to study ligand/receptor interactions in two systems: the nicotinic acetylcholine receptor (nAChR) and the retinoic acid receptor (RAR). The nAChR study details how a combination of homology modeling, molecular dynamics (MD), blind docking, and free energy analysis may be used to determine the binding site of a ligand with few clues from experiment to guide the search. Specifically, the binding site for a class of negative allosteric nAChR modulators was successfully identified. The computationally predicted binding site was verified by functional assays performed on receptors that were mutated at the suspected binding site. Additionally, a comparison of structural data from homologous proteins and MD simulations of the receptor in complex with an allosteric modulator lead to a proposed mode of allosteric antagonism that involves inhibition of C loop closure, thereby preventing channel opening. In the RAR study, both computation and experiment were applied to characterize the activity of two ß-apocarotenoids that have been previously described as antagonists of all-trans retinoic acid (ATRA), the endogenous RAR agonist. The activity of RAR ligands is related to how they influence the interaction between the receptor and coactivator proteins that lead to gene transcription. The results of isothermal titration calorimetry (ITC) experiments indicate that the ß-apocarotenoids induce an interaction between the receptor and coactivator that is intermediate in strength between the unliganded and ATRA-bound receptor, indicating that these compounds would be most accurately characterized as partial agonists instead of antagonists. One of the partial agonists, ß-apo-13-carotenone, exhibits an unexpectedly high affinity for RAR given its chemical differences from known high-affinity binders. Modeling this compound in the RAR binding site lead to the hypothesis that a covalent interaction may be occurring between the carotenone and a conserved cysteine residue in the binding pocket. While not conclusive, NMR and mass spectrometry experiments suggest that this interaction is indeed occurring. Computational free energy analysis was also performed between the ligand-bound receptors and the coactivator. Using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method applied to microsecond MD simulations, very strong correlation was achieved between the computational binding energies and the experimental ITC data, providing support that the compounds were correctly modeled in the RAR binding pocket. Converged binding energy averages that lead to the strong correlation with experiment were contingent upon simulation lengths of ~1 µs, and inclusion of both the calculated PBSA free energy of solvation and entropic components of binding were found to strengthen the correlation. Finally, Chapter 5 includes a study on the parameterization of a new atom type for use in the pair-wise additive AMBER force field. The sulfonium atom type is not included in the current set of parameters since it is relatively uncommon in biology. However, S-adenosylmethionine (SAM), the most common methyl donor in biology, is a notable exception. The development of sulfonium parameters required for the MD simulation of SAM is discussed in detail.
Chenglong Li (Advisor)
Charles Bell (Committee Member)
Michael Paulaitis (Committee Member)
354 p.
Biophysics
computational chemistry; molecular modeling; free energy analysis; biophysics; protein receptors; docking; force field parameterization; SAM; nAChR; RAR; retinoic acid receptor; nicotinic acetylcholine receptor; S-adenosylmethionine

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Citations

  • Pavlovicz, R. E. (2014). Investigation of Protein/Ligand Interactions Relating Structural Dynamics to Function: Combined Computational and Experimental Approaches [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397220613

    APA Style (7th edition)

  • Pavlovicz, Ryan. Investigation of Protein/Ligand Interactions Relating Structural Dynamics to Function: Combined Computational and Experimental Approaches. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1397220613.

    MLA Style (8th edition)

  • Pavlovicz, Ryan. "Investigation of Protein/Ligand Interactions Relating Structural Dynamics to Function: Combined Computational and Experimental Approaches." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397220613

    Chicago Manual of Style (17th edition)

osu1397220613
1,090
© 2014, some rights reserved.Creative Commons License Investigation of Protein/Ligand Interactions Relating Structural Dynamics to Function: Combined Computational and Experimental Approaches by Ryan Elliott Pavlovicz is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.