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Development of Chemical Biology Probes to Regulate and Study the Phase Separation Mechanism of Biomolecular Condensates

Passos, Chrysoula Christie (2024) Development of Chemical Biology Probes to Regulate and Study the Phase Separation Mechanism of Biomolecular Condensates. Master's Thesis, University of Pittsburgh. (Unpublished)

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From lava lamps to oil droplets in water, the process of liquid-liquid phase separation informs the world we live in. Biologically, liquid-liquid phase separation involves components of similar properties associating and forming droplets in cells, formally termed “biomolecular condensates”. While eukaryotic cells organize their biochemistry through both membrane-bound organelles and membrane-less organelles, prokaryotes can also compartmentalize their cellular components through membrane-less organelles. These biomolecular condensates form to regulate rates of reactions, sequester components from the surrounding milieu, and scaffold other protein-protein interactions. We have been investigating the process of liquid-liquid phase separation in prokaryotes, specifically in bacteria, and we have examined a bacterial endoribonuclease called RNase E, which is known to phase separate through liquid-liquid phase separation and mediate mRNA processing and decay. While the stepwise mechanism by which RNase E phase separates currently remains under investigation, it is suggested that multivalent, electrostatic protein-protein interactions play a critical role in the protein’s self-assembly. As a result of these strong yet reversible short-range interactions, multivalent bonding can lead to the oligomerization of protein into spherical phase-separated droplets. A recent framework developed to help us better visualize these multivalent protein interactions is the “Stickers and Spacers” model proposed by Rohit Pappu. In this model, multivalent proteins and RNA molecules can be thought of as scaffolds that drive intracellular phase transitions. Along an associative protein polymer, one can identify areas with attractive interactions between aromatic, charged, or polar residues termed “stickers.” These stickers represent interacting regions within the intrinsically disordered proteins that contribute to phase separation, while intermediary interactions weaker in strength to “stickers” can be identified as “spacers". We aim to use this model and framework to help us better understand the phase separation mechanism of RNase E by which we may identify potential small molecule inhibitors that have the potential to impact RNase E droplet morphology and kinetics, as these may ultimately lead to a novel class of antibiotics.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Passos, Chrysoula Christieccp30@pitt.educcp30
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorChilders, W. Sethwschild@pitt.eduwschild
Committee MemberWeber, Stephensweber@pitt.edusweber
Committee MemberIslam, Kabirulkai27@pitt.edukai27
Date: 8 May 2024
Date Type: Publication
Defense Date: 15 December 2022
Approval Date: 8 May 2024
Submission Date: 28 March 2024
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 54
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Biomolecular condensates
Date Deposited: 08 May 2024 17:44
Last Modified: 08 May 2024 17:44


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