ORCID

https://orcid.org/0000-0003-1525-2553

Date of Award

Winter 12-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Protein aggregation is common in biological processes including neurodegenerative diseases, as well as artificial development in biopharmaceutical production and protein storage. Therefore, understanding proteins involved in aggregation systems is important. It is generally difficult for high resolution structural methods, such as X-ray or NMR, to interrogate proteins possessing aggregation propensities. On the other hand, techniques that tolerate solution heterogeneities, such as circular dichroism, only provide global or low-resolution information. Mass spectrometry (MS)-based methods are appropriate and powerful in such applications because they can deal with mixtures. Additionally, primary sequence, post-translational modification, and structural information can be obtained at low sample consumptions and fast turnover. This thesis aims to develop and apply MS-based methods in understanding three proteins involved in two aggregation/oligomerization systems. Bacteria biofilm provide protection for bacteria from host defense and in severe environments, causing great concerns in human health. The major proteinaceous component of biofilm, Curli, is the first system on which we focus. Curli family has seven members. We focus on the major curli subunit CsgA and a proposed CsgA chaperon, CsgE, in chapter 2 to 4. CsgA is an amyloid protein that shares common features with other well-known disease-associated amyloid proteins. CsgE also forms oligomers, for which the details were unknown when we started the project. In chapter 5 to 7, we concentrate on apolipoprotein E (apoE), which is has strong implications in Alzheimer's disease. ApoE regulates lipid and cholesterol transportation in the blood and central nervous system. There is no structure of wild-type apoE as it exists as a mixture of monomer, dimer, and tetramer at micromolar concentration in vitro. The primary method used in the thesis is hydrogen-deuterium exchange (HDX), which is described in detail in chapter 1. Principles of instrumentation and fragmentation are also included. In chapter 2, we first applied conventional, continuous HDX to identify the oligomerization interface of CsgE. This result guided a mutagenesis study for developing a monomeric mutant, whose NMR structure now has been published. We also compared the difference between the monomeric mutant and the wild-type protein by continuous HDX. We found an interesting structural rearrangement for wild type CsgE by using pulsed HDX. In chapter 3, we improved the pulsed HDX platform, adding a reference peptide, to study the aggregation behavior of CsgA. The addition of reference peptide allows us to monitor CsgA aggregation from another dimension and helps HDX data interpretation. We systematically followed CsgA deamidation by collision-induced dissociation (CID) and MS label-free quantification as described in chapter 4. We discovered that deamidated CsgA loses its ability to form fibrils by using the pulsed HDX platform developed in chapter 2, circular dichroism, and the Thiofavin T fluorescence assay. In chapter 5, we characterized the binding between a small molecule compound and apoE. A sequential digestion was implemented in HDX for better coverage and shorter peptides (higher HDX resolution). We extended our understanding of the same system, using two adapted HDX-based techniques, in chapter 6. Peptide-level binding affinities are extracted by using PLIMSTEX, and unfolding properties are monitored by using peptide-level SUPREX. In chapter 7, we employed native MS to inspect oligomeric composition and study the gas-phase structures. With ion mobility data and additional support from electron-capture dissociation, we built a coarse-grained model of the tetrameric complex of apoE. Conclusions and future directions are discussed in chapter 8. The six research chapters demonstrate the power of MS in understanding protein structure, protein-protein/ligand interaction and oligomerization/aggregation. These approaches can be extended to other biological systems that are difficult to study by conventional analytical methods. Every method provides information from a unique perspective. To advance our understanding of complicated systems, multiple techniques, including MS, should be used in combination to achieve complementary information and the most complete results and interpretation.

Language

English (en)

Chair and Committee

Michael L. Gross

Committee Members

Carl Frieden, John-Stephen Taylor, Gary Patti, Alexander Barnes

Comments

Permanent URL: https://doi.org/10.7936/K71Z42T9

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