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Developing an efficient binding assay to quantify molecular interactions with the profilin protein

Hughson, Alexandra (2023) Developing an efficient binding assay to quantify molecular interactions with the profilin protein. Undergraduate Thesis, University of Pittsburgh. (Unpublished)

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After eye injuries, aberrant blood vessel growth, known as pathological ocular angiogenesis, can cause vision loss or blindness. This same pathological angiogenesis can also contribute to unregulated cell proliferation related to cancers. Previous research in animal models has shown that compounds that block actin-Pfn1 interactions can inhibit harmful angiogenesis. Native polyacrylamide gel electrophoresis (PAGE) offers an inexpensive, quick, and quantifiable assay system for screening interactions between Pfn1, actin, and anti-angiogenic C74 compounds. Unfortunately, wild type Pfn1 (WT-Pfn1) does not run into the gel on most native PAGE buffering systems since it has very little charge. To resolve this issue, a variant of profilin called profilin-3E (3E-Pfn1) is designed, adding three negatively charged residues to its N-terminus. 3E-Pfn1 is shown to be visualized on native PAGE, proving the viability of the assay. To prove the utility of 3E-Pfn1, it must be demonstrated that it functions biochemically and biophysically like WT-Pfn1. To do this, a protein thermal shift (PTS) assay is used to demonstrate that 3E-Pfn1 has a similar melting temperature to WT-Pfn1, and therefore would be biochemically similar. Likewise, structural modeling and crystallization techniques, previously employed on WT-Pfn1, are used to validate that 3E-Pfn1 is physically like that of the WT. In the future, native PAGE and PTS assays will be used to demonstrate that 3E-Pfn1 maintains polyproline binding as a further validation method. Once validated, 3E-Pfn1 can be utilized to quickly screen many future variants of C74 that are currently under development.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Hughson, Alexandraakh50@pitt.eduakh500009-0001-7071-8478
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorVanDemark, Andrewandyv@pitt.eduandyv0000-0003-3424-4831
Committee MemberAmbrose, Zandreazaa4@pitt.eduzaa40000-0002-7518-7418
Committee MemberKoes, Daviddkoes@pitt.edudkoes0000-0002-6892-6614
Committee MemberMacbeth, Markmrmacbet@butler.edu0000-0002-8610-2766
Date: 15 April 2023
Date Type: Publication
Defense Date: 16 March 2023
Approval Date: 15 April 2023
Submission Date: 3 April 2023
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 45
Institution: University of Pittsburgh
Schools and Programs: David C. Frederick Honors College
Dietrich School of Arts and Sciences > Biological Sciences
Degree: BPhil - Bachelor of Philosophy
Thesis Type: Undergraduate Thesis
Refereed: Yes
Uncontrolled Keywords: profilin, actin, native PAGE, SDS PAGE, protein purification, crystallography, angiogenesis, cancer
Date Deposited: 15 Apr 2023 18:10
Last Modified: 15 Apr 2023 18:10


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