Transition from Soluble to Insoluble Glucose Storage Polymers in Biological Systems

Researchers from Iowa State University are testing potential molecular mechanisms that convert soluble glucan storage, i.e., glycogen, to semi-crystalline starch. The primordial form of carbohydrate storage is the soluble polymer glycogen, which is found in many prokaryotic species and eukaryotes including fungi and animals. Chloroplast-containing species, i.e., green algae and land plants, in contrast, store glucans in insoluble granules. At some point in earth’s history, glycogen metabolism was modified so that glucan polymers became semi-crystalline and assembled into insoluble granules. This event greatly increased photosynthetic carbon assimilation by eukaryotes and was a requisite step in the spread of land plants on earth. Granular starch further facilitated the subsequent rise of our agronomic system that relies primarily on seeds of cereal crops. Thus, the mechanisms that converted glycogen metabolism into starch metabolism are a fundamental component of our biosphere. The research team will generate in vitro and in a reconstituted synthetic biology system a “carbohydrate chaperone”, which is a protein complex that stimulates crystallization of branched glucan polymers. Discoveries from the experimental system could impact biotechnological applications of polymer science. Structural design of proteins for use in the experimental system will be performed by secondary school students. The proposed glucan chaperone consists of the proteins ISA1 and ISA2 in a heterotetrametric complex with a highly elongated structure extending for 230 Å. Eight glucan binding sites are organized over the length of the complex, and this organization is proposed to bring linear chains into proximity and so stimulate formation of double helices, which are favored entropically. In addition to this function, the non-mutant complex catalyzes branch linkage hydrolysis. The binding sites will be varied in recombinant complexes which will be purified and tested for affinity to various glucans. The variant complexes also will be tested for stimulation of soluble branched glucan crystallization in a synthetic biology system hosted in yeast cells. A primordial ISA protein from a prokaryote that functions in branched glucan catabolism will be evolved into a plant form that instead functions as a glucan chaperone, thus testing a hypothesis for how starch arose during establishment of photosynthesis in eukaryotes. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.