Starch in plants is a large polymer of glucose that stores excess glucose for times of limited photosynthesis. Starch is used in many industrial processes such as laundry and papermaking. In plants, regulation of starch storage and degradation is a multifaceted system that allows the plant to adjust carbohydrate storage in response to a variety of environmental and developmental conditions. However, we are unsure how this process is regulated. β-amylases (BAMs) hydrolyze starch to produce maltose. Plants contain many BAM proteins and possibly each has a unique function and regulatory mechanism, although this is an area of active research. The degradation of starch by BAM1 is activated when the plant undergoes osmotic stress. BAM1 is suggested to be regulated by a redox reaction, however, the precise regulatory mechanism is unclear although it is thought to involve disulfide formation between an N-terminal cysteine and another within the amylase domain. A better understanding of the structure and the activity of BAM1 will allow us to improve many crucial industries. To address the structure of BAM1, we purified the protein from E. coli cells and ensured that the protein was folded using circular dichroism. We then collected small-angle X-ray scattering (SAXS) data on BAM1 and analyzed the data to determine solution dimensions of BAM1. Those data were then compared against SAXS data of other BAM proteins collected in our lab. The preliminary SAXS data and comparison support BAM1 being a monomeric protein with an extended N-terminus. Another part of this project was to determine the activity of BAM1 and whether the activity is affected by oxidation. We are optimizing the Bicinchoninic acid (BCA) assay, as well as a maltose-binding protein green fluorescent protein (MBP-GFP) assay to investigate the activity of BAM1 and the effects of oxidizing conditions. Further investigation into the disulfide bond will be done using DSF, and CD.
Support or Funding Information
This work was supported by a National Science Foundation Research at Undergraduate Institutions grant to JDM and CEB (MCB-1932755). Additional support comes from the National Institutes of Health project ALS-ENABLE (P30 GM124169) and a HighEnd Instrumentation Grant (S10OD018483).
This work was supported by a National Science Foundation Research at Undergraduatelt;brgt;Institutions grant to JDM and CEB (MCB-1932755).amp;nbsp;Additional support comeslt;brgt;from the National Institutes of Health project ALS-ENABLE (P30 GM124169) and alt;brgt;HighEnd Instrumentation Grant (S10OD018483).
