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Measurements of Protein Folding/Misfolding using Pulsed Electron Spin Resonance

Jun, Sangmi (2010) Measurements of Protein Folding/Misfolding using Pulsed Electron Spin Resonance. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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We describe a new electron spin resonance (ESR) method that measures interspin distances at physiological conditions. The method is used to examine the melting of a polyalanine peptide, which is of considerable current interest in the field of protein folding. One end of the peptide is tagged with a Cu(II) ion and the other with a nitroxide spin label. The rapidly relaxing electron spin, Cu(II), enhances the electron spin flip rate of the nitroxide. This change in the relaxation rate depends on the interspin distance, r. As the peptide unfolds, the interspin distance changes. The ESR determined interspin distance decreases from the folded to unfolded state whereas the MD simulation shows an increased distance of the peptide in the PPII geometry. Therefore, the ESR results indicate that the polyalanine peptide does not melt into an extended PPII structure in the unfolded state. The ESR results also show that the change in interspin distances is clearly in concordance with the change in helicity and demonstrates an important application of the pulsed ESR method to monitor unfolding transition at physiological temperatures in biomolecules.In the second part of this research, the local environment of the Cu(II) ion in amyloid-¥â peptide (A¥â) is investigated by electron spin echo envelope modulation ESR spectroscopy. The aggregation of A¥â is implicated in the pathogenesis of Alzheimer¡¯s disease. Distinct differences in coordination of Cu(II) to A¥â are observed as the Cu(II) concentration increases. The overall morphology of A¥â aggregates, shown by transmission electron microscopy and atomic force microscopy images, also depends on the Cu(II) concentration. At low concentration of Cu(II), A¥â fibrils are observed and Cu(II) is coordinated by N-terminal histidine residues. As the concentration of Cu(II) increases, the images suggest a significant increase in the proportion of granular amorphous aggregates, and ESR spectra indicate a second copper-binding site that exists in a proton-rich environment. The results strongly suggest that the misfolding mechanism of A¥â depends on the Cu(II) concentration. This research significantly enhances our understanding of the misfolding mechanism in A¥â, and elucidates the relationship between the microscopic Cu(II)-A¥â interaction and macroscopic structure.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Jun,, sangmi96@gmail.comSAJ22
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSaxena, Sunilsksaxena@pitt.eduSKSAXENA
Committee MemberWilcox, Craigdaylite@pitt.eduDAYLITE
Committee MemberWaldeck, Daviddave@pitt.eduDAVE
Committee MemberRule, Gordon
Date: 28 January 2010
Date Type: Completion
Defense Date: 9 December 2009
Approval Date: 28 January 2010
Submission Date: 10 December 2009
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Alzheimer¡¯s disease; Amyloid-¥â peptide; Copper-binding site; Distance measurement; Electron spin resonance
Other ID:, etd-12102009-083600
Date Deposited: 10 Nov 2011 20:10
Last Modified: 15 Nov 2016 13:54


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