Tae Kim , '14

Date of Award

Spring 2014

Document Type

Restricted Thesis

Terms of Use

© 2014 Tae Kim. All rights reserved. Access to this work is restricted to users within the Swarthmore College network and may only be used for non-commercial, educational, and research purposes. Sharing with users outside of the Swarthmore College network is expressly prohibited. For all other uses, including reproduction and distribution, please contact the copyright holder.

Degree Name

Bachelor of Arts

Department

Physics & Astronomy Department

First Advisor

Catherine Hirshfeld Crouch

Second Advisor

Carl H. Grossman

Third Advisor

Kathleen P. Howard

Abstract

M2 protein of Influenza A virus is a multifunctional membrane bound protein integral to the viral life cycle. To study the dynamic atomic level details of how M2 induces membrane curvature, electron paramagnetic resonance (EPR) spectroscopy can be used to study the conformation of the M2 protein in cholesterol containing membrane bilayers. When using EPR spectroscopy, spin label reporter groups are attached to specific sites of M2. For results to be reliable, the addition of a spin label should not affect the overall shape and the function of the protein.

In order to test that M2 retains its functionality upon spin labeling, a method to assay the curvature generating properties of M2 protein was developed to control for possible functional perturbation due to site directed spin label (SDSL). In this functional assay, M2 is reconstituted into LUVs by means of detergent-mediated reconstitution, and the resulting proteoliposomes are then electroformed into GUVs using the partial dehydration protocol. In this work, the detergent-mediated reconstitution protocol was optimized by using DLS to determine optimal detergent concentration, and the partial dehydration protocol was optimized to minimize ILV production.

The final step of the functional assay involves counting ILVs in a control sample without M2 and in a sample including M2. Using the manual assay, the differences between the control and the sample with M2, in the functional assay, was statistically insignificant. In order to increase the statistical significance of the differences between two samples, large number of GUVs needed to be counted. To aid counting large number of GUVs, an image analysis program was created to automatically analyze the confocal microscopy images of GUVs. With the improved statistics that are made possible by this automated analysis, the functional assay can distinguish the samples with and without M2, and thereby confirm the curvature generating function of M2.

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