Examining post-translational modifications in alpha-synuclein through semi-synthesis and single molecule spectroscopy / Buyan Pan.

Format:

Book

Thesis/Dissertation

Author/Creator:

Pan, Buyan, author.

Contributor:

Petersson, E. James, degree supervisor.

Rhoades, Elizabeth, degree supervisor.

University of Pennsylvania. Department of Chemistry, degree granting institution.

Language:

English

Subjects (All):

Chemistry.

Biophysics.

Pathology.

Chemistry--Penn dissertations.

Penn dissertations--Chemistry.

Local Subjects:

Chemistry.

Biophysics.

Pathology.

Chemistry--Penn dissertations.

Penn dissertations--Chemistry.

Genre:

Academic theses.

Physical Description:

1 online resource (283 pages)

Contained In:

Dissertations Abstracts International 83-03B.

Place of Publication:

[Philadelphia, Pennsylvania] : University of Pennsylvania ; Ann Arbor : ProQuest Dissertations & Theses, 2021.

Language Note:

English

System Details:

Mode of access: World Wide Web.

text file

Summary:

Alpha-synuclein (αS) is an intrinsically disordered protein believed to mediate synaptic vesicle trafficking through interactions with lipid surfaces. The pathological, fibrillar aggregates of αS are implicated in a class of neurodegenerative disorders including Parkinson's disease. A variety of post-translational modifications (PTMs) are known or hypothesized to affect both the function of αS and its propensity to form fibrils. In this work, we introduce PTMs of interest along with fluorescent probes into αS through semi-synthesis, affording site-specifically modified αS for functional and disease-relevant studies. Organic and peptide syntheses, protein expression using unnatural amino acids, and semi-synthetic strategies involving native chemical ligation (NCL) are optimized for the incorporation of challenging or novel modifications such as phosphorylation, arginylation, and acetylation. Where applicable, we evaluate trade-offs between the synthetic ease of using a mimic and the extent to which it recapitulates the properties of the authentic PTM. We characterize the aggregation of PTM-bearing αS and identify the kinetic and thermodynamic effects on fibril formation brought about by subtle changes in levels of modification. A bidirectional effect on aggregation is observed for tyrosine-39 phosphorylated αS, whereas a dose-dependent reduction in fibril formation is seen for arginylated αS, with additive effects for arginylation at more than one site. Single molecule Forster resonance energy transfer (smFRET) measurements reveal changes in monomer conformation that underlie a possible mechanism for the differential aggregation profile of phosphorylated αS. In addition to its disease-relevant characteristics, we examine the function of modified αS using fluorescence correlation spectroscopy (FCS) to assay its affinity for lipid vesicles. We find that neither phosphorylation nor arginylation alters lipid binding. However, we observe by smFRET that phosphorylation gives rise to conformational rearrangements in the presence of lipid vesicles. Finally, we elucidate the molecular basis of interactions between αS and the human acetyl transferase enzyme NatB, complemented with investigations at the single molecule level into the dynamics of a synthetic inhibitor consisting of full-length αS. Our results shed light on the substantial impact that subtle changes in PTM levels have on protein structure, native function, and fibril formation in disease.

Notes:

Source: Dissertations Abstracts International, Volume: 83-03, Section: B.

Advisors: Petersson, E. James; Rhoades, Elizabeth; Committee members: Molander, Gary ; Marmorstein, Ronen; Shorter, James.

Department: Chemistry.

Ph.D. University of Pennsylvania 2021.

Local Notes:

School code: 0175

ISBN:

9798535593562

Access Restriction:

Restricted for use by site license.

This item is not available from ProQuest Dissertations & Theses.

This item must not be sold to any third party vendors.