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Title:Structural investigations of membrane-interacting proteins using nuclear magnetic resonance spectroscopy and other biophysical techniques
Author(s):Ojoawo, Adedolapo Moyinoluwa
Director of Research:Rienstra, Chad M
Doctoral Committee Chair(s):Nair, Satish
Doctoral Committee Member(s):Pogolerov, Taras; Jin, Hong
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):NMR spectroscopy
Molecular Dynamics
Protein structure
Membrane
Blood coagulation
gold nanoparticles
alpha-synuclein
Abstract:Structural information of protein is vital for understanding its function as well as its interactions with membrane and other proteins. Aberrant protein expression or disruption in protein-protein/protein-membrane interactions lead to disease formation. Membrane-interacting proteins play critical roles in a wide range of biological processes and their dysfunction contribute to a number of diseases including blood coagulation disorders and neurodegenerative diseases. In addition, many membrane-interacting proteins are targets for drugs. Understanding their structure will be beneficial for probing interactions with other proteins and membrane as well as provide insights into their function. This thesis focuses on the structural studies of protein in different environments. In particular, the biophysical and structural properties of membrane-interacting proteins are studied using a range of biophysical methods such as solution nuclear magnetic resonance (NMR) spectroscopy, magic-angle spinning (MAS) solid-state NMR spectroscopy (SSNMR), dynamic light scattering (DLS), and molecular dynamics (MD). There are many biophysical techniques used for protein structure characterization such as X-ray crystallography, NMR, and emerging ones such as cryo-electron microscopy (cryo-EM). Due to the complexity of biological system, a single technique isn’t often enough to obtain a detailed picture of the structural and dynamic properties of a biological molecule. Experimental chemical shifts from solution and solid-state NMR methods provide structural information on a biological molecule at the atomic level. However, for large proteins with disordered regions, it is often difficult to obtain complete structural detail with just NMR because complete resonance assignments and large amounts of distance restraints are needed to achieve this, thus making the structure refinement/characterization costly and time-consuming. In X-ray crystal structures, disordered regions or loops are either missing or characterized with high thermal factor. Also, protein structure refinement programs or molecular dynamics simulations without real experimental data cannot provide accurate structural models of protein. Tissue factor protein studied here is an example, with X-ray crystal structures of its extracellular domain missing electron density in functionally relevant regions. To refine the structure of the extracellular domain (soluble) of TF (sTF), we employed the integration of complementary data from NMR with X-ray crystallography using CamShift, a method which readily converts chemical shifts to interatomic distances for use as structural restraints in molecular dynamics simulation of protein, to perform rapid chemical shift-based structure refinement. Chemical shifts contain enough information to determine protein structure at high resolution. They are very sensitive to the local environment of a protein, thus using them as structural restraints in MD simulations would enable mapping of conformation changes in proteins in different iii environment such as in the presence of ligand or lipid membrane without the need for extensive additional data collection such as NOEs or other distance restraints measurements. Here, uniformly (U)-13C, 15N labeled sTF were prepared in solution for solution NMR studies and in ammonium sulfate precipitated (microcrystalline) form for MAS-SSNMR studies. Multidimensional NMR experiments were acquired to obtain complete resonance assignments of sTF in both forms. In addition, (U)-13C, 15N labeled sTF mutants, G90A and S85T were prepared and studied using NMR to aid assignments of residues near mutation sites. X-ray crystal structures of sTF are lacking electron density in functionally relevant dynamic loops accessible by NMR. Using CamShift, each set of chemical shift assignments were used to drive the X-ray crystal structure to refined structures that agrees with the experimental chemical shifts. The structures were validated by calculating the chemical shifts from the final models and comparing to experimental data. Structural comparisons between the crystalline and solution form of sTF were made focusing on the unresolved loop regions missing in the X-ray structure. We observe a major change in the conformation of the loop P79-P92 in sTF as compared to the X-ray crystal structure and minor conformational difference in the membrane interacting loops. Another protein investigated with a different set of biophysical techniques in this thesis is alpha-synuclein (α-syn), a protein primarily found in the presynaptic terminals of nerve cells. Its aggregation and fibrillation are the cause of neurodegenerative diseases like Parkinson’s. Specific point mutations to the amino-acid sequence of α-syn including A30P and E46K, are linked to development of hereditary Parkinson’s Disease. α-syn is an intrinsically disordered protein that studies have shown to be capable of inducing physical changes in membranes containing anionic phospholipids. This feature of remodeling membranes poses a challenge when studying the size-dependence of α-syn interactions with phospholipid vesicles. To investigate the role of vesicle surface curvature in α-syn binding behavior, rigid SDS-coated gold nanoparticles (AuNP) were used as lipid membrane mimics to investigate the binding interaction of WT α-syn and two of its mutants (A30P and E46K). The binding interactions with SDS-coated AuNP of varying sizes were studied using DLS and proteomics.
Issue Date:2020-07-17
Type:Thesis
URI:http://hdl.handle.net/2142/108702
Rights Information:Copyright 2020 Adedolapo Ojoawo
Date Available in IDEALS:2020-10-07
Date Deposited:2020-08


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