Identifier

etd-11162006-233014

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

Protein aggregation occurs under many circumstances, from the dynamic assembly of tubulin to form microtubules, the aggregation of actin into filaments, as well as plaque formation by amyloid precipitation. One important requirement in studying the mechanism of amyloid aggregation is the ability to monitor the growth kinetics over a wide range in size scales (10 nm to microns) with time that spans microseconds to seconds. Understanding the mechanisms of the aggregation may then lead to improved design of drugs to help control or suppress the aggregation process. In this dissertation, the physical characterization of the â-amyloid peptide and its interaction with áá-amino acid peptide-based mediators was investigated from the early, rapidly evolving stage to the later, slowly diffusing peptide stage by the application of fluorescence photobleaching recovery (FPR). The diffusion rates of â-amyloid peptide and â-amyloid assemblies under the effects of variables including concentration of â-amyloid, blocker peptide, ionic strength, pH, time and temperature were accessible by this method. In some instances, dynamic light scattering (DLS), which acquire signals greater than 10 decades of lag times, without requiring a dye label was used for comparison and to account for the limitations of any given approach. Attachment of 5-carboxyfluorescein did not affect the integrity of the protein and the measured diffusion coefficients were similar to those measured by diffusion ordered spectroscopy (DOSY) and from theoretical expectations. FPR proved more sensitive than DLS for detection of low oligomer aggregates of the â-amyloid peptide coexisting with much larger fibrils. We were able to reverse the conformation of the peptide from the low oligomeric state to the aggregated state under neutral and acidic pH conditions and confirmed that the peptide growth increased with increasing ionic strength. The interaction of â-amyloid peptide with membranes results in several membrane-perturbing effects which may play a pivotal role in the pathogenic cascade leading to Alzheimer’s disease. Model phospholipid bilayer membranes consisting of 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) and 1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] dodecanoyl]-sn-Glycero-3-Phosphocholine (18:1-12:0 NBD PC) were prepared on mica. FPR proved to be a useful technique for obtaining information of the nature of membrane fluidity upon interaction with the â-amyloid peptide.

Date

2006

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

Paul S. Russo

Included in

Chemistry Commons

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