SusChEM: Carbohydrate Recognition in Type B Carbohydrate Binding Modules

Intellectual Merit. Production of fuels and chemicals from renewable biomass in response to increasing societal energy demands will play a key role in ensuring energy security and slowing climate change. Accordingly, understanding the mechanisms by which enzymes break down biomass is of primary importance to enhanced biotechnology developments for improved deconstruction. Enzymatic hydrolysis of biomass is usually conducted with multi--].modular cellulase enzymes consisting of a catalytic domain and one or more carbohydrate binding module (CBM) domains connected by peptide linkers. A significant majority of ongoing studies aimed at improving biomass conversion technology focus exclusively on functionality within the catalytic domain. However, the often--].overlooked CBMs play significant roles in modulating function and cannot be discounted given mounting evidence suggesting CBMs are highly functional protein domains. Furthermore, CBMs have demonstrated functionality in biotechnology beyond biomass conversion including cell immobilization, molecular targeting, and as molecular probes for mapping the still--].undefined plant cell wall glyco--].architecture. In--].depth molecular--]. level investigation of the complex solid and soluble carbohydrate substrate recognition mechanisms in CBMs promises to achieve enhanced kinetics and economics in biomass conversion technology. The proposed research focuses on uncovering the mechanisms behind carbohydrate recognition in Type B CBMs, one of three types classified by structural and functional similarities. Type B CBMs are thought to selectively bind oligomeric chains up to 6 monomers long. However, a subset of cellulose--]. specific CBM families (4, 17, and 28) has been shown to bind to non--].crystalline cellulose more tightly than oligomers and in a manner that discriminates between surface topology. These CBMs also often appear in tandem, harnessing multivalent interactions to enhance affinity. Biochemical studies have painted a broad picture of Type B CBM carbohydrate recognition. However, little is known about how structural and dynamical features contribute to substrate binding, with some findings not explained by structure and binding alone. The proposed work will address the hypothesis that Type B CBM carbohydrate recognition mechanisms are subtly different for oligomeric and non--].crystalline substrates, which may account for the evolutionary occurrence of tandem binding modules. The following objectives will test this hypothesis: 1. Identify the origins of oligomeric carbohydrate recognition within and across Type B CBMs. Molecular dynamics simulations and free energy methods will be used to evaluate the molecular--]. level origins of oligomeric carbohydrate recognition within and across the three cellulose--].specific Type B CBMs. Residues important to substrate binding will be mutated using advanced thermodynamic cycles to assess their role in tight binding. An advanced sampling free energy method will be used to calculate absolute binding free energies. 2. Examine the origins of non--].crystalline carbohydrate recognition in Type B CBMs. Two Type B CBMs, from families 17 and 28, will be modeled over a non--].crystalline cellulose substrate using molecular dynamics simulations. Alchemical free energy pathways will be used to determine the free energy of binding to non--].crystalline cellulose. 3. Relate carbohydrate recognition mechanisms to avidity and cooperative binding in tandem Type B CBMs. Molecular dynamics simulations of a tandem CBM construct in solution and in proximity to a representative non--].crystalline cellulose substrate will be used to relate findings from the individual modules to the tandem construct behavior. Principle component analysis will elucidate allostery contributing to cooperative binding and avidity. Broader Impacts. Beyond the technological benefit to society . sustainable and affordable alternative energy, the proposed research will be integrated with aggressive research infrastructure development through a statewide outreach program intended to encourage and inform researchers of all levels about the existing supercomputing resources available. The latter activity is intended to reach underrepresented groups in rural Kentucky through online access to information, rather than exclusively classroom--].based instruction. The PI also has a proven track record in encouraging K--].12 girls to explore STEM careers and will incorporate the proposed research into her local outreach efforts.