BASIL2022GV3R8E

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Abstract

The Protein Data Bank (PDB) contains approximately 188 thousand protein structures, 5000 of which have not been assigned a specific function. As a part of the Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) project, we were tasked with analyzing and determining the function of one of these proteins, PDB ID 3r8e. This protein is a putative kinase, which is of interest due to the key roles kinases play in many cellular processes. Utilizing the modules the BASIL consortium provides, a series of in silico and in vitro experiments were conducted. The 3r8e protein was first studied using a variety of in silico tools, including BLASTp, Pfam, and DALI. Based on our in silico results, glucose was determined to be the most likely substrate for 3r8e and was used for further in vitro characterization of the protein. To confirm the in silico function prediction for the 3r8e protein, bacterial protein overexpression, affinity chromatography purification, and coupled kinase activity assays were utilized. Multiple sugar substrates for 3r8e were tested, including glucose. The coupled kinase assay results confirmed that 3r8e likely plays a role in glucose phosphorylation, aligning with our in silico conclusions. Future directions include repeating the above experiments and testing additional hexose substrates to further characterize the enzymatic activity of 3r8e. Overall, we have strong preliminary evidence that the 3r8e protein is a glucose kinase and future work will allow us to confirm this.

Introduction

Like many proteins with solved crystal structures, protein 3r8e has an uncharacterized and unconfirmed function. Extracted from and being a part of a bacteria that is found in soil, no one knows what the role of this specific protein plays in relation to the bacteria. Apart of a research project, our group set out to solve the function of this protein to then potentially provide further insight of the protein's relationship to the bacteria. Below is the general workflow of how we got our results.

Methods

Experimental Results/Function

Project Implications

Conclusions/Future Direction

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