Abstract The human gut microbial community influences many aspects of human physiology via the output of short chain fatty acids from the fermentation of dietary carbohydrates. Ruminococcus bromii, a keystone species in the human gut, degrades dietary resistant starch (RS) and cross-feeds other gut bacteria that produce butyrate, a short chain fatty acid with potent anti-inflammatory and anti-tumorigenic properties. R. bromii has an amylosome, a complex of secreted starch-active proteins, that is predicted to be responsible for its unique ability to degrade resistant starch (RS). However, the molecular determinants of the amylosome that confer RS degradation are poorly understood. The amylosome is predicted to be assembled via calcium- dependent protein-protein interactions. Via proteomic analysis, I identified Doc20 as one of the most abundant proteins enriched from EDTA-washed R. bromii. Doc20 is comprised of two starch-binding domains separated by an extended Thr/Pro-rich linker followed by a dockerin domain, a calcium-dependent protein-protein interaction domain that facilitates incorporation into the amylosome. While we have a theoretical framework by which R. bromii assembles via dockerins, the nature of the discrete interactions between amylosome components and molecular features of these proteins are unknown, preventing a mechanistic understanding of how R. bromii degrades RS. I have solved the crystal structures of domain 1 and a close homolog of domain 2 of Doc20 to reveal that these are starch-binding domains. Despite these data being informative of the domains of Doc20 in isolation, how the starch-binding capabilities of Doc20 may be influenced by the linker and how these domains interact are unknown. I discovered that Doc20 binds to Sca5, a cell wall-anchored protein that has two starch-binding domains. I aim to structurally analyze Doc20’s binding interface with Sca5 and define Doc20’s interaction network with other amylosome proteins to understand how Doc20 contributes to the amylosome. In some multiprotein systems that employ a dockerin-mediated protein interaction network, flexible linkers of dockerin-containing proteins contract after binding to their target protein. I aim to characterize the molecular dynamics of the linker region that separates domains 1 and 2 of Doc20 by using small angle x-ray scattering (SAXS) and solve the crystal structure of the Doc20-Sca5 complex. I also aim to elucidate the protein interaction network of Doc20 over time. My expected outcome is to create a temporal, molecular-scale map of the starch-binding network of proteins that interact with the abundant amylosome protein Doc20. These results are expected to have a positive impact as they will uncover the biochemical details of Doc20 within the context of the amylosome which will provide essential insight into RS degradation by gut bacteria.