Faculty Sponsor: Colin Smith
Tihitina Gebeyehu
Tihitina Gebeyehu is a rising junior (’27) from Addis Ababa, Ethiopia, double majoring in Chemistry and Economics. On campus, she is involved in the International Student Advisory Board, and in her free time, she enjoys hanging out with her friends, beading, and crocheting.
Abstract: Computationally designed miniproteins could be used in medicine as effective therapeutic drugs. They are better substitutes for monoclonal antibodies due to their reduced side effects. They have smaller binding sites, diverse shapes, and possible biological functions. However, despite their promising properties, there is still much to be understood about structural heterogeneity in the proteins, which can limit their efficacy. To address the issue, we use EHEE_rd2_0005 as a model protein to manipulate structural heterogeneity in miniproteins. Temperature-dependent NMR and replica exchange molecular dynamics simulations have revealed significant structural and chemical heterogeneity in the model. Previous work showed flipping of tryptophan 35 on the intermediate timescale. On a slow timescale, deamidation of asparagine 31 was observed, and at high temperatures, transient unfolding occurred, accompanied by an increased rate of deamidation. Using the wild-type EHEE_rd2_0005 and mutants, we analyzed gNhsqc peak volumes to assess the impact of specific mutations on the W35 side chain. Three of the six mutants (E11P, V28R, and D8P) showed more high-volume peaks than the wild-type, suggesting that these mutations may stabilize the W35 flip. This work aims to advance our understanding of how different single-point mutations affect the structural dynamics of the protein and to identify mutations that favor one W35 rotamer without disrupting the protein’s secondary structure.
Additionally, we analyzed Circular Dichroism (CD) melting data for three computationally designed miniproteins being studied by Benjamin Levine to assess their thermal stability and unfolding. The experimental results will then be compared to their computational unfolding simulations.
Tihitina_Gebeyehu_QAC_Summer_2025_Poster