Designed Expanded Co-Crystals for Guest Structure Determination

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Nontechnical DescriptionTo understand biology and to design medical interventions, it is essential to observe the structure adopted by large biological molecules. One of the main methods for determining those structures is to coax the molecules into arranging themselves into a crystal, and then to collect the diffraction pattern that results when these crystals are exposed to an X-ray beam. This conventional process has a high failure rate. Researchers have therefore long sought to develop scaffold crystals that can organize guest molecules.This project will produce a new class of candidate scaffold crystals. Technically, these new crystalline superstructures are co-crystals, since they are composed of both protein and DNA building blocks. To identify the most promising materials in this family, the team will use computational design and all-atom simulations to evaluate candidates for experimental validation. These new crystal materials will feature a modular design where “DNA struts” with any desired sequence are exposed to open channels that are large enough to permit the movement of guest proteins. The research team will grow crystals in which these DNA struts have sequences that are specifically recognized by certain DNA-binding proteins. When these guest proteins latch onto their target DNA they will join the crystal lattice and become observable via X-ray diffraction. This approach circumvents the haphazard crystal growth process that is the basis for conventional crystallography. The resulting materials may therefore provide a transformative method for scientists to routinely and easily observe atomic details for the complexes between DNA and DNA-binding proteins, complexes that drive critically important life processes such as transcription and regulation.Broader Impacts: Precise control of the 3-D position of functional molecules within a scaffold crystal opens the door for materials with unprecedented performance for diverse additional applications including biosensing, catalysis, energy conversion, biomedicine, and biotechnology. To partially explore these alternative applications, the team will provide mentorship and funding for 3 years of undergraduate-led biomolecular design teams (2020, 2021, and 2022), with each project culminating in the international BIOMOD competition. Inspiring and training the next generation of students to innovate at the biomaterials design frontier will directly accelerate the pace of discovery, to the benefit of the scientific community and the nation.Technical DescriptionThe team will use atomistic modeling and simulation to design “expandable” protein:DNA co-crystal structures. The approach is pragmatic: re-engineering protein-DNA systems that are already known to form co-crystals. Inserted DNA struts will be tuned to preserve existing crystallographic contacts and symmetry. Co-crystals will be grown, optimized, and stabilized using crosslinking and ligation chemistry. Critically, large solvent channels in the resulting co-crystals will permit post-crystallization additive molecular assembly, using the inserted DNA struts with the appropriate sequence for site-specific capture of cognate DNA-binding molecules. This approach circumvents the haphazard nucleation and growth that underlies conventional crystallography. The team will determine if DNA-binding molecules captured by the scaffold crystal become visible via X-ray diffraction. Goal 1. Design candidate engineered co-crystals. Use all-atom simulations to prioritize variants for experimental validation.Goal 2. Express, purify, and crystallize novel designed crystals composed of engineered protein and DNA building blocks. Optimize crystal growth by varying the length and sequence of the DNA blocks.Goal 3. Stabilize designed co-crystals using chemical crosslinking. Capture a guest protein that binds specifically to the DNA sequences that were inserted into the struts.The proposed materials have unique aspects that warrant investigation as a potential platform technology. [1] The design and experimental validation of highly porous protein:DNA co-crystals as scaffolds is unknown, as is [2] the subsequent site-specific capture of DNA-binding proteins therein. Unlike conventional biomolecular crystals, the proposed scaffold crystals [3] have pore sizes that may be expanded by changing the number of base pairs present within DNA struts, and [4] have topologies that are amenable to modular incorporation of arbitrary DNA sequences. This project will focus on the structural biology application of these materials, but side applications (e.g. biosensing or catalysis) may be the focus of the 3 annual undergraduate BIOMOD biomolecular design teams for whom the project will provide mentorship and funding.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.