Control and functionalization of protein nanofibrils using genetic and chemical modification to define assembly mechanisms and design principles

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This research program seeks a better understanding of protein aggregation for applications in food quality, safety and human health. Protein amyloid fibrils, or `nanofibrils', are a highly organized type of protein aggregate associated with disease (e.g., Alzheimer's, ALS & mad cow) in certain cases, while in other cases they play a functional role (e.g., bacterial biofilm, barnacle adhesion, and human melanin synthesis). A mechanistic understanding of protein nanofibril formation could be used both to prevent unwanted aggregation (i.e., disease or bacterial biofilm formation) and to develop novel biomimetic materials and devices, such as catalytic arrays, biosensors, cell-scaffolds, bioelectronics, and controlled-release of bioactive compounds. These may find applications in food processing (enzyme immobilization for bioreactors and biosensors), tissue engineering (cultured meat for food; cultured tissues for medicine) or as functional foods (controlled release of bioactives). Protein nanofibrils have several unique properties, including a high aspect ratio (=1000:10 nm), strength, and stability, a unique surface chemistry, and the ability to self-propagate. A major challenge is that the design rules for building protein nanofibrils are not well known. Proteins can be induced to self-assemble into nanofibrils, threads of protein a few molecules in width and 1000's in length, in a complex process that follows different pathways, involving protein unfolding, misfolding, oligomer formation, proto-fibril and mature fibril formation. A second challenge is that utilization of protein nanofibrils for creating functional materials requires the ability to chemically modify proteins site-specifically, selectively, and in a manner compatible with the fibril structure. Targeting specific locations on a protein generally cannot rely on naturally abundant functional groups (e.g., amine, carboxylic acid and sulfhydryl groups), as these are present at multiple locations and their reactivity is limited to certain pH and redox conditions. To overcome these limitations, new protein engineering tools will be used to genetically insert `click' chemical groups into specific sites of model proteins to allow for chemical labelling in a well-defined, efficient manner. These click groups are inert to biological molecules and react only with a specific partner, which will be used to conjugate the nanofibrils with other functional molecules (e.g., peptides, enzymes, carbohydrates, lipids, DNA, nanoparticles, fluorescent dyes). Biophysical tools will be used to examine the effects of such genetic and chemical modifications on fibril assembly, structure and functionality. This platform will be used to examine the mechanism of fibril assembly and to create new nanoscale materials and devices (e.g., biosensors, ordered enzyme-arrays, & materials for controlled release) that ultimately may help enhance food quality and safety.