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Publication Date

2024-5

First Advisor

Nathan D. Derr

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

biochemistry, protein design, molecular motors, yeast, chimeric proteins

Abstract

Motor proteins, such as cytoplasmic dynein, play a vital role in the maintenance of spatial, temporal, and metabolic homeostasis within eukaryotic cells. These proteins transport cargo utilizing the cell’s highway-like network of microtubules. The goal of this project is to design chimeric proteins between S. cerevisiae (brewer’s yeast) and H. sapiens (human/mammal) by using genetic manipulation to alter the microtubule binding domain (MTBD) and create the following chimeras in yeast: 1. fully mammalian dynein, 2. Mammalian MTBD with point mutation A3337G, 3. fully yeast MTBD within a mammalian dynein, and 4. yeast MTBD with point mutation G3337A. The focus of this research is to decipher the mechanisms of processivity in cytoplasmic dynein. During this process a plasmid was successfully assembled containing the gene sequence of a uracil (URA3) cassette and dynein flanking regions using NEB’s HiFi technology. An additional cassette with truncated human dynein was designed and ordered from GenScript. PCRs were performed to isolate DNA fragments to be used in transformations further along in the project Chimeric motors can be observed via total internal reflection fluorescence microscopy (TIRF), via single motor assays or ensemble assays in flow chambers or immobility/motility assays. The immobility/motility assay procedure has been another focus of this project, which allows for the observation of the entire ATP driven stepping cycle, which will help distinguish how these chimeras affect dynein processivity by identifying non-processive motors and non-binding motors.

Rights

©2024 Emily Swindell. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

Comments

96 pages: color illustrations, charts. Includes bibliographical references.

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