Author: Chloe Hutchison Major: Biology
Gel Electrophoresis for Visualizing Tubulin Reconstitution
My name is Chloe Hutchison, and I am a Junior Biology Major. My current and future research in the lab will be characterizing the binding interactions of ruthenium based polypyridyl complexes with Microtubules. Microtubules (MT) play a crucial role in cell cycle division by separating duplicated chromosomes through their dynamic instability. In previous research, a small molecule called RPC2 has shown to stabilize this dynamic feature of MTs therefore disrupting their function which can lead to apoptosis. Taxol is a current chemotherapeutic agent which exploits this mechanism by acting as a Microtubule Stabilizing Agent clearly showing the importance of further work with this small molecules and others with similar structure.
I joined Dr. Adams Biochemistry lab in Fall of 2020 and began learning the fundamental techniques involving protein expression and purification. This involved protein expression using E. Coli which had a genetically engineered plasmid which contained our protein of interest. I expressed CDC42, used a His-Tag HPLC column to extract only the CDC42 and then confirmed its purity through SDS-PAGE Gel Electrophoresis. Once I had mastered these techniques I was ready to begin my work on my Honors Thesis in Spring 2021.
Dr. Adams’ lab has largely focused on small molecule binding interactions with proteins that are important in modulating cell growth and differentiation. Dr. Adams specifically has been working in collaboration with a Professor from the University of Texas who provided us with the small molecule of interest, RPC2. I proposed to use a new method for this system called Intrinsic Tryptophan Fluorescence to calculate the dissociation constant Kd and thus binding affinities of our small molecule and the Tubulin protein. I purchased my protein kit and reconstituted the components. Within the proposal and time frame of starting the experiments, new small molecules, DP and DB, were sent to our lab and were expected to be water soluble. This was important as the RPC2 compound required DMSO for dissolution and limited the cellular applications of this molecule. We ran a solubility test on the DP and DB small molecules, and they were soluble in water, which shifted the focus of my research on these new molecules.
I began preparing my protein in concentrations for the Fluorescence titrations for each tubulin subunit from the 3mg/mL stocks I had prepared from the reconstitution which was used in a 1 x10 dilution. I ran 4 titration experiments, 2 for each small molecule with different concentrations of DB/DP, to optimize the conditions; I chose to move forward with 1mM of the small molecule. I ran triplicate Fluorescence Titrations for each small molecule, starting with 400uL of tubulin and titrating 5uL of small molecule 10 times. The FL at 330nm was measured, plotted and fit to the Hill Plot equation which gave the slope as the Kd. The Kd for the DP molecule was calculated to be 1.561×10-7 M and Kd for the DB molecule was 1.91×10-7 M. The hyperbolic curve and Kd in the micromolar range show that these molecules are binding to the MTs and binding in ranges similar to other RPCs.
Throughout this process of starting my Honors Thesis work, I have been faced with many challenges. One of the most difficult things I had to overcome was starting. With such a vast amount of literature I struggled narrowing down how to build a system. I remember meeting with Dr. Adams, and I asked him what my plan should be and how should I start, and he replied “Chloe, in my lab I will not hold your hand, but I will walk beside you.” He has challenged me to go beyond what I am comfortable with, ask thoughtful questions to understand why and not what and to take accountability for my own personal success and for that I am exceptionally grateful.
The Honors College grant made this research possible; I was able to purchase a protein kit with a purity >99.9% which allotted me more time to work on the experimentation rather than protein expression and purification. My time working in lab was alongside Djamali Muhoza, a Senior Graduate Assistant in our lab. He spent many hours teaching me the Fluorescence Titrations techniques, how to optimize the conditions and interpret the data and mastering the computer systems to analyze my data. His long hours and help has allowed me to grow in my independence and I will carry that knowledge into my future work over the next year, so a big thank you to him. In the next semester, I plan to continue the experiments with the DB/DP small molecules and MTs. I will work on titrations at different temperatures that will allow me to extract thermodynamic parameters that will further characterize these binding interactions. I am extremely thankful for the Honors College for funding and to Dr. Adams and Djamali Muhoza for their continued support and encouragement.