Structural and functional characterization of n-terminally acetylated alpha-synuclein Emily Brackhahn

Format:

Book

Thesis/Dissertation

Author/Creator:

Brackhahn, Emily, author.

Contributor:

University of Pennsylvania. Chemistry., degree granting institution.

Language:

English

Subjects (All):

Biophysics.

Biochemistry.

Chemistry.

0786.

0487.

0485.

Local Subjects:

Biophysics.

Biochemistry.

Chemistry.

0786.

0487.

0485.

Genre:

Academic theses

Physical Description:

1 online resource (163 pages)

Contained In:

Dissertations Abstracts International 87-12B

Place of Publication:

Ann Arbor : ProQuest Dissertations and Theses, 2026

Language Note:

English

Summary:

α-Synuclein (αSyn) is an intrinsically disordered protein whose conformational flexibility underlies both its proposed physiological roles at synaptic membranes and its pathological aggregation in Parkinson's disease. Although αSyn is constitutively N-terminally acetylated in vivo, most biophysical studies have relied on unmodified protein, leaving the structural and functional consequences of this modification incompletely defined. In addition, αSyn is subject to regulated post-translational modifications such as phosphorylation at Ser129, whose effects have largely been examined in isolation. This dissertation investigates how N-terminal acetylation, alone and in combination with Ser129 phosphorylation, modulates αSyn's conformational ensemble and membrane-binding behavior. Using a rational protein engineering strategy, we generated a panel of αSyn variants with systematically altered N-terminal chemistry, including unmodified, acetylated, asparagine-substituted, and succinylated forms. Circular dichroism and solution NMR spectroscopy were used to quantify how these modifications tune transient N-terminal helicity without inducing stable folding. Functional consequences were assessed using fluorescence correlation spectroscopy to measure membrane binding across vesicles of defined composition, charge density, and curvature. These experiments reveal that N-terminal acetylation enhances αSyn membrane affinity through increased local helicity and hydrophobic anchoring, while alternative N-terminal chemistries differentially modulate binding in a membrane-dependent manner. To examine the combinatorial effects of post-translational regulation, we developed a co-expression strategy enabling site-specific incorporation of both N-terminal acetylation and Ser129 phosphorylation. Comparative analyses reveal that phosphorylation partially attenuates the acetylation-induced enhancement of helicity and membrane binding, yielding an intermediate structural and functional phenotype. Finally, cell-based methodologies were developed to enable future investigation of αSyn in a biological context. These include an assay for extracellular αSyn uptake and the implementation of fluorescence correlation spectroscopy in living cells. While the cellular measurements presented here are preliminary, these approaches establish a foundation for extending the biophysical insights gained in this work to cellular systems. Together, this work establishes a mechanistic link between αSyn's biochemical state, conformational ensemble properties, and membrane binding function

Notes:

Source: Dissertations Abstracts International, Volume: 87-12, Section: B.

Advisors: Rhoades, Elizabeth Committee members: Christianson, David W.; Baumgart, Tobias; McCallum, Monica E.

Ph.D. University of Pennsylvania 2026

Vendor supplied data

Local Notes:

School code: 0175

ISBN:

9798247981015

Access Restriction:

Restricted for use by site license