Dubyna, Jason2023-11-152023-11-152023-11-032023-11-03http://hdl.handle.net/1993/37791Hexokinase and VDAC are two critical proteins throughout fungi. Despite the considerable number of studies on VDAC-hexokinase interactions in animal systems, little research has been attempted looking at their interaction in fungal systems. The existing work described in the literature suggests that yeast hexokinase and VDAC have no interaction, unlike in mammalian and plant systems. However, previous research used crude methods to test binding interactions and it has not been re-examined until recently, when Ferens et al. (Ferens et al., 2019) used detergent-solubilized Neurospora crassa VDAC and Saccharomyces cerevisiae (yeast) hexokinase to demonstrate that hexokinase isoform 1 (Hxk1) was capable of binding and hexokinase 2 (Hxk2) was not. The current work sought first to overexpress and purify the hexokinase isoforms from yeast to confirm that hexokinase 1 binds to VDAC and hexokinase 2 does not. This was achieved and binding affinities were determined using Microscale Thermophoresis. Hxk1 has a binding affinity for VDAC of 31.6 +/- 15.6 μM, whereas Hxk2 did not bind. The second goal was to determine the region of hexokinase 1 that is responsible for the isoform-specific binding. The two isozymes are very similar except for a few discrete regions. Variants were created by swapping short amino acid sequences from the Hxk1 binding isoform into the Hxk2 isoform. Hxk25-34, in which residues 25-34 were replaced with those from Hxk1, had a binding affinity close to that of Hxk1. Therefore, altering one small region of Hxk2 imparted binding affinity to VDAC. Our work opens potential avenues into investigating the functions of fungal VDAC-hexokinase interactions involved in gene regulation during glucose repression in yeast, a process in which hexokinases are involved.engHexokinaseVDACVoltage Dependent Anion Channelmaster thesis