Date of Award
Chemistry and Biochemistry
Michelle K. Knowles
Lipid membranes play a vital role in cell signaling processes. Membrane shape and lipid content affect interactions between cellular membranes and proteins. This research focuses on characterizing those interactions and their impact by using various biochemical and biophysical assays. These assays were applied to C-reactive protein (CRP), an immune system protein that interacts with lipid membranes and has at least two forms with different properties. Native, pentameric CRP (pCRP) is found in blood serum and is commonly used as a marker for inflammation. The modified form of CRP (mCRP) binds to the protein C1q, which activates the complement immune response. There is a gap in our knowledge of the biochemical information for the two forms of CRP and several different methods are used to make mCRP in the lab. In this work, we analyzed the pCRP to mCRP conversion using three biochemical denaturants: guanidine hydrochloride, urea with ethylenediaminetetraacetic acid (EDTA), and heating with dilute sodium dodecyl sulfate (SDS). After comparing results from biochemical assays to determine structural and functional differences, mCRP treated with dilute SDS and heat was the form that both significantly bound C1q and had an intrinsic tryptophan fluorescence level as anticipated for an intermediate form.
After characterizing biochemical differences between functional mCRP and native pCRP forms, binding of pCRP and mCRP to curved membranes was analyzed with an in vitro membrane mimic assay that starts with a nanoscale pattern on a glass coverslip to create curvature for a supported lipid bilayer to coat. By using fluorescent nanoparticles of a specific size, curvature based interactions can be visualized and quantified. Results show that the amount of CRP binding is not only dependent on protein conformation but also on membrane curvature size.
Expanding the membrane curvature assay, used to visualize CRP membrane binding, to allow for cellular experiments was achieved by first melting the nanoparticles and then coating them with silicon dioxide, creating a permanent nanoparticle patterned substrate. Custom chambers were created and added to the nanoparticle patterned coverglass to reduce the required volume. Characterizations of the silicon dioxide coated surface were done with analysis of fluorescent lipids in supported lipid bilayers. The assays and results from this research not only contribute to better biochemical knowledge of CRP, but will be useful for future studies of proteins involved in cellular processes that have membrane interactions.
Moon, Carrie Leine, "Identifying the Relevance of C-Reactive Protein Conformers and the Advancement of a Membrane Curvature Binding Assay" (2017). Electronic Theses and Dissertations. 1266.
Recieved from ProQuest
Carrie Leine Moon