Date of Completion

Spring 5-4-2018

Thesis Advisor(s)

James Schaff; Johann Peter Gogarten

Honors Major

Molecular and Cell Biology

Disciplines

Bioinformatics | Cell Biology | Computational Biology | Investigative Techniques | Laboratory and Basic Science Research | Molecular Biology | Systems Biology

Abstract

Computational modeling of cell-cell interactions can grant clues and can answer questions about an experiment, especially for observations about binding interactions and kinetics. This approach was used to investigate an interaction between a light-oxygen-voltage (LOV) domain and an engineered protein called Zdark (Zdk). The LOV domain is membrane-bound while Zdk is cytosolic. The LOV domain and Zdk bind strongly in dark (Kd 26.2 nM), and weakly upon exposure to blue light (Kd > 4 μM). Total internal reflection fluorescence (TIRF) images are acquired of Zdk, the fluorescent species bound to a mCherry tag, and the loss of fluorescence is observed upon illumination. However, secondary binding sites of transfer-messenger RNA SsrA and stringent starvation protein B SspB can impact the off rate. SsrA is an RNA binding protein that is bound to the LOV molecule, and SspB, which enhances recognition for the SsrA binding site, is bound to the Zdk peptide. The affinity between the two binding sites alters the kinetics of the dissociation of the LOV domain and Zdk. To note, the SsrA and SspB binding sites can still be connected when the LOV domain and Zdk dissociate.

Modeling of this experiment was primarily done through Virtual Cell. VCell is a free computational tool for modeling and simulation of various cell biology systems that can be used to perform and analyze simulations. This was used to mathematically model the microscopy experiment, with goals of gaining new insight to the underlying biology and evaluating quantitative analysis of the experimental data. Several important tasks include gathering theoretical values for the kinetics of the systems in terms of the on rate and off rate and looking for observations about the interaction of the secondary binding sites. This will all be used in an attempt to construct an experimental protocol utilizing this optogenetic system, especially for lower affinity binding.

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