Date of Award

1-1-2016

Document Type

Dissertation

Degree Name

Ph.D.

First Advisor

Michelle K. Knowles, Ph.D.

Second Advisor

Susan Sadler

Third Advisor

Sandra S. Eaton

Fourth Advisor

John Latham

Fifth Advisor

Bryan J. Cowen

Keywords

ANS, 8-anilino-1-naphthalenesulfonic acid, Apotosis, CRP, C-reactive protein, LysoPC, Lysophosphatidylcholine, mCRP, Membrane curvature, Modified CRPP, Modified c-reactive protein

Abstract

C-reactive protein (CRP) is a serum protein that binds to damaged membranes and initiates the complement immune response. Different forms of CRP are thought to alter how the body responds to inflammation and the degradation of foreign material. Despite knowing that a modified form of CRP(mCRP) binds to downstream protein binding partners better than the native pentameric form, the role of CRP conformation on lipid binding is yet unknown. In this work, three main assays were performed to characterize how conformation affects CRP-membrane interactions. The first assay utilized supported lipid bilayers that mimic the plasma membrane of apoptotic cells. The results show that CRP interactions vary with protein conformation, lipid composition, and membrane shape and that the mechanism by which CRP recognizes damaged membranes depends on the combination of all three. The second assay focused on CRP interactions with cellular membranes by using induced apoptosis in MES-SA cells to characterize how CRP recognizes damaged cellular membrane. Monitoring the disassociation of pCRP to mCRP on apoptotic cell membrane with a fluorescent RNA aptamer that specifically binds mCRP but not pCRP allowed for quantitative analysis. Thirdly, a fluorescence quenching assay was utilized to characterize CRP conformational states using 8-anilino-1-naphthalenesulfonic acid (ANS), a fluorescence probe that binds to the hydrophobic regions of a protein. The unfolding of CRP using different denaturants was assessed using tryptophan fluorescence assay. Urea-EDTA, Guanidine-HCl, and SDS with heat were used to perturb the pentameric state. All treatments give rise to a monomeric state in native PAGE experiments. As revealed by ANS fluorescence, treatment with 2.5M GndHCl, 3 or 6 M urea, or 0.01% SDS caused hydrophobic portions of CRP to be exposed. Based on this data, we conclude that the two forms of CRP studied here have different mechanisms of action.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Aml Abd Alhamed Alnaas

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

121 p.

Discipline

Chemistry, Biochemistry

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