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Characterizing conformation ensembles of proteins and peptides by combining spectroscopic techniques
Dissertation   Open access

Characterizing conformation ensembles of proteins and peptides by combining spectroscopic techniques

Bridget Milorey
Doctor of Philosophy (Ph.D.), Drexel University
Mar 2021
DOI:
https://doi.org/10.17918/00000551
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Abstract

Chemistry, Physical and theoretical Biophysics
The once central dogma of protein biophysics stated that protein function is determined by structure. There is now an enormous body of evidence disproving this notion, including the discovery of intrinsically disordered proteins and the existence of multi-functional proteins. In part I of this thesis, random coil models that were once used to predict the structures of unfolded or disordered proteins are contested. Intrinsically disordered proteins (IDPs) and regions (IDRs) are often rich with charged amino acid residues. Several lines of experimental and even computational evidence suggest that polypeptides and proteins that carry high net charges tend to adopt extended conformations, and the average end-to-end distance tends to exceed expectations for self-avoiding random coils. Herein, charged arginine (R) residues in glycine (G) capped GRRG and GRRRG are shown to induce nearest neighbor interactions (NNIs) that stabilize extended structures. In a second study of GXXG, GXXXG model peptides, protonated aspartic acid (D) was studied in GDDG and GDDDG. D is known to show a high intrinsic preference for turn-like structures, which may be important sites for early protein folding events. The results presented here indicate that repeating D sequences show strong NNIs in that turn-like structures are stabilized. In part II of this thesis, the binding interaction of the heme protein cytochrome c and liposomes containing the anionic phospholipid cardiolipin is studied for both the reduced and oxidized species of the protein. The reduced protein is not the subject of much literature, so this binding study filled an important gap in knowledge. The protein was found to bind, unfold, and then oxidize on the liposome surface. The binding process for the oxidized species of the protein was studied at slightly acidic pH, where a different binding mechanism from that at neutral pH was probed. The protein unfolding on the liposome surface led to population of a high-spin species, which may have important implications for the protein gaining peroxidase activity and initiating apoptosis (organized cell death).

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