EAGER: Coherent Guest Protein Organization Inside Host Protein Crystals

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With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Snow at Colorado State University, is developing a new approach for observing proteins in atomic detail. Structure determination of proteins is a key bottleneck for understanding and engineering protein molecules. The project aims to circumvent the challenges of growing conventional protein crystals, by taking the unprecedented step of attaching target proteins to specific sites within pre-existing, crystalline scaffolds. Once guest proteins are attached to the scaffold crystal, they need to adopt a structure to be visible via X-ray diffraction. This is a highly risky step but the pay-off will be huge too. Success will open the door for an accessible and economical alternative to conventional structure determination methods for protein studies.The current dominant method for structure determination in atomic detail is X-ray crystallography, but this method requires a brute force search through non-physiological solution conditions, looking for the ?needle-in-a-haystack? condition in which the target protein crystallizes. Unfortunately, despite exhaustive screening, most proteins of interest (~70%) do not form crystals. Other proteins are difficult to obtain in sufficient quantities to make the attempt. Finally, the crystals that do grow reveal a structure adopted under artificial conditions, a single snapshot that dramatically under-represents protein mobility. The motivational insight for this project is the recognition that materials diffract X-rays if they consist of a highly-ordered, repeating lattice, but that the lattice need not be composed only of target protein. Supported by this EAGER grant, Professor Snow is tryong to demonstrate that guest proteins anchored to the crystalline scaffold can adopt a coherent structure visible via X-ray diffraction and then correlate the guest structure observed as a function of solution conditions. To achieve this goal, the Snow group first prepare thiol bearing scaffold crystals and covalently install guest proteins using disulfide bonds or homobifunctional crosslinkers. The resulting crystals are screened for coherent guest organization via solvent substitution and x-ray diffraction. When necessary, the scaffolds are further engineered via chemical modification or genetic mutation. The project provides an educational training opportunity in cutting-edge areas of bionanotechnology, chemical biology, applied biophysics, and molecular modeling.