Determining the Binding Affinities of Starch‐Protein Interactions using Concanavalin based Sedimentation Assay

Abstract

Starch is ideally suited as an energy storing molecule in plant systems because of its unique structure and biophysical properties. However, these same properties limit the accessibility of hydrolyzing enzymes that break down starch at night. Phosphorylation of the glucosyl residues of the outer layers of the starch granule by dikinases makes the starch granule partially water soluble and accessible to starch degrading enzymes such as amylases. The subsequent removal of phosphate groups by glucan phosphatases is necessary for complete starch hydrolysis. Despite their important biological roles, our understanding of how dikinases, glucan phosphatases and amylases interact with the complex starch granule is not complete. In biological context, determining the affinities of starch–protein interactions are challenging due to low binding affinities and the heterogeneity and complexity of the structure of starch. Using a concanavalin based in vitro sedimentation assay, the goal of this project is to develop a novel binding assay to measure the affinities of starch ‐protein interactions. We determined the Kd app for amylopectin and glucan phosphatase Starch Excess4 and compared that to currently existing methods to determine the validity of the method. The further optimization of our method will allow us to determine binding affinities of glucan phosphatase, dikinase and amylases families of enzymes.