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Title:Membranous sterol dynamics and interactions with amphotericin B
Author(s):Della Ripa, Lisa Anne
Director of Research:Rienstra, Chad M.
Doctoral Committee Chair(s):Rienstra, Chad M.
Doctoral Committee Member(s):Burke, Martin D.; Chan, Jefferson; Mitchell, Doug A.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Solid-State NMR
molecular dynamics
cholesterol
amphotericin B
Abstract:Sterols are ubiquitous membrane-active molecules that regulate the fluidity cellular membranes. Specifically, cholesterol (Chol) plays an active role in modifying the functionality of the membrane proteins. Chol is vital for cell function, as it is essential to a myriad of biochemical and biophysical processes. Furthermore, Chol is involved in numerous important processes such as regulation of membrane properties and signaling events. However, the exact mechanism or structural understanding of Chol’s role in these events is not always well resolved. Here we utilize solid-state NMR (SSNMR) to probe the atomistic interactions of Chol with membranes and other related systems. Collaboration with synthetic and computational groups has enabled a powerful multidisciplinary approach to study these systems using SSNMR, biosynthesis, molecular dynamics (MD) simulations and quantum mechanics (QM) calculations. The atomistic details of Chol's interactions with phospholipids and proteins are of fundamental interest, and spectroscopic methods that interrogate these properties at high resolution remain an attractive area of research. Towards this end, here we describe a high-yielding biosynthesis of 13C-labeled Chol and magic-angle spinning (MAS) SSNMR approaches to examining the structure and dynamics of Chol in lipid bilayers. We quantify the incorporation levels of 13C enrichment and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements throughout the entire molecule. Additionally, with highly-enriched 13C-labeled Chol, we obtained these measurements in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methodologies to study sterols and their interactions with drugs, lipids and proteins. The long-standing challenge of an accurate description of membranous Chol dynamics at atomistic resolution has been addressed by decades-long efforts, but is still not completely resolved. In particular, the structure, dynamics and interactions of the middle portion of Chol are largely undescribed because 2H or 13C-Chol are commercially available with labeling only at the head or tail. A novel combination of new SSNMR experiments, computational MD and QM methods captured dynamics and the chemical environment of uniformly-labeled 13C Chol. The agreement between experiment and computation led to the determination of the tail conformational ensembles present in the membrane. The molecular motions of Chol appear to be coupled to specific tail conformations. Our integrated experimental and computational approach enables the detection of changes in sterol dynamics, which can be used to elucidate important interactions with other membrane agents. Using 3D SSNMR experiments that recouple 1H-13C dipolar couplings and employ a through-bond scalar mixing, MD simulations of Chol in lipid bilayers and QM calculations, we additionally aim to understand the key interactions that determine the sterol-specificity to Amphotericin B (AmB), a powerful antifungal drug used to treat life threatening fungal infections. AmB forms large aggregates that are neither crystalline nor soluble, so structural information is difficult or impossible to obtain via x-ray crystallography or solution-state NMR. We have recently determined that AmB kills yeast cells primarily by binding to ergosterol and forming a large extramembranous sterol sponge. This model hypothesizes that interactions of AmB with ergosterol determine its ability to kill yeast, whereas binding to Chol is responsible for toxicity in human cells. SSNMR spectroscopy is uniquely able to detect and quantify the binding of sterols to AmB in the sterol sponge at atomistic detail. Results from these studies will provide an avenue to an improved therapeutic index for this drug. Additionally, we hope to expand the use of this toolkit to atomically-resolve the putative binding sites of several membrane proteins.
Issue Date:2019-05-21
Type:Text
URI:http://hdl.handle.net/2142/105741
Rights Information:Copyright 2019 Lisa A. Della Ripa
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08


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