Performed a PAGE to ensure protein stability before experimentation

Author: Chloe Hutchinson | Major: Biology | Semester: Spring 2022

My name is Chloe Hutchison and I just finished up my last semester with Dr. Adams laboratory and successfully finished and defended my Senior Honors Thesis. My project was focused on characterizing the binding interactions between Novel-Ruthenium Polypyridyl Complexes and Microtubules. Microtubules (MT) play a crucial role in mitotic events by separating duplicated chromosomes through their dynamic activity, cycling through rounds of polymerization and depolymerization. Previously published research identified tris-chelate Ruthenium based compounds as interacting with MTs in vitro and in vivo with a mechanism of action by stabilizing the polymerized state, halting the cell cycle and leading to apoptosis. Taxol is a current chemotherapeutic agent which exploits this mechanism by acting as a Microtubule Stabilizing Agent (MSA) clearly showing the importance of further work with novel small molecules within this subcategory and others with similar structures.

I first reached out to Dr. Adams after attending his presentation at the Arkansas Summer Research Institute in July 2020. His lab largely focused on on small molecule binding interactions with proteins that are important in modulating cell growth and differentiation, which tied in my undergraduate interests and future goals of pursuing a career in medicine. By having a foundation and experience with biochemical systems and regulatory mechanisms, I developed such a greater breadth of understanding of many physiological systems implicated in human disease.

Dr. Adams has been working in a collaborative research effort and our laboratory was provided with novel RPC’s, ([Ru(dip)₂bpy]Cl₂) (DB) and ([Ru(dip)₂phen]Cl₂ (DP), which were synthesized in efforts to make more water-soluble. I ran solubility studies and identified they were water soluble and soluble in highly concentrated stocks of 10mM. These compounds are devoid of organic limitations in comparison to previously studied hydrophobic RPCs which is an unique feat for potential drug therapeutics. I also ran fluorescence titrations and identified these novel small molecules bind to MT’s and are binding in the micromolar range which is in agreement with other known MSA’s.

This semester I started off by performing a polymerization assay which measures the kinetics and degree of polymerization. I used Tubulin alone as the negative control in these experiments and Paclitaxel (Taxol) + Tubulin as the positive control as it is a current MSA used in antitumor therapies. The experiment was run at 37^oC to reduce the impact of temperature on the rates of polymerization. Once I normalized the data, I fit the polymerization curves to the Hill Plot Model to extract the kinetic parameters. The V_max for this experiment represents the maximal rate at which polymerization is occurring. The MT + Paclitaxel with a V_max=0.79OD/min acts as the positive control as Paclitaxel is a current MSA used in the treatment of cancer. The MT alone acts as the negative control with a V_max=0.22OD/min. There is approximately a 3-fold enhancement of the rate of polymerization in the presence of Paclitaxel supporting its identity as a known MSA. DP was found to have a V_max=0.43 OD/min, which is about a 2-fold increase in the rate of polymerization, suggesting DP in vitro has microtubule-stabilizing properties. DB was found to have a V_max=0.56 OD/min which is 2.5 faster than the MT alone, suggesting DB also has microtubule-stabilizing properties. The total OD corresponds to the length of the polymer, which showed at saturation, these novel RPCs (DP and DB) also enhance the degree of polymerization with respect to the MT alone, further suggesting their identify as an MSA.

I also had the opportunity to run an Isothermal Titration Calorimetry binding experiment between DP and MTs, at 37⁰C, to extract thermodynamic parameters. These results are as follows: ΔG=-8.86kcal/mol, ΔH= -14.6 kcal/mol, -TΔS=5.6 cal/mol, K_d=0.6uM for the DP:MT binding interaction. This is within excellent agreement with thermodynamic parameters of published Ruthenium-Polypyridyl Complexes that act as MSAs in vivo and would suggest they have similar modes and mechanisms of binding, supporting further research with this novel system.

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 Dr. Adams lab was alongside Djamali Muhoza and Emilio Duverna, alumni graduate assistants. They spent many hours teaching me the different techniques, how to optimize my parameters and how to use software to dissect and analyze my results. Their long hours and help have allowed me to grow in my independence and I will carry that into my next academic endeavor, a research fellowship at the NIH in the Department of Transfusion Medicine. And the biggest thank you to Dr. Adams for believing in me and challenging me each semester to grow in my scientific approach. His support has gone above and beyond what I could have expected in a Principal Investigator and will have a lasting impact on my career and future goals.