Coiled Coil-mediated Assembly of Protein Nano-cages and their Application to Bio-catalysis
Abstract
Protein-based compartments are intimately involved in cellular functions, where they serve as storage vessels, assist in protein folding and protein degradation, and in some cases contain enzymatic reactions. Inspired by Nature, there is great interest in both designing protein cages de novo and re-engineering natural protein cages for wide-ranging applications including targeted drug delivery, polyvalent display of antigens, in vivo imaging, templating of nano-particles, and encapsulation of enzymes in protein nano-reactors. Designed self-assembling protein systems are particularly attractive as building blocks for ÒsmartÓ biomaterials that exploit the rich structural and functional properties of proteins, while potentially overcoming the limitations of natural protein cages. We have developed a simple, modular design strategy for assembling protein cages in which protein assembly is mediated by short, parallel, coiled coil domains. These are genetically fused to a larger, symmetrical building block protein through a short, flexible spacer sequence. By using combinations of symmetry axes that are unique to the geometry of the desired protein cage, we have shown it is possible to assemble well-defined protein cages using symmetry considerations alone. This approach greatly simplifies the design process and, other than symmetry, places few constraints on the proteins that can be used as building blocks. In this proposal we aim to apply our design strategy to tackle the following problems: Design protein cages with new geometries We will examine whether our methodology can be extended generate new quaternary structures, including those that Nature has not evolved, by using our coiled coils to assemble cages from other proteins that possess different quaternary structures and rotation symmetries e.g. dimeric (C2) tetrameric (C4), pentameric (C5) and hexameric (C6) proteins. Stabilize enzymes through assembly A remarkably stable icosahedral cage that we designed points to the potential of protein assembly to stabilize enzyme activity; a property that if generalizable would be of considerable practical utility. Importantly, it was not necessary to alter the structure of the enzyme to stabilize it: rather, the thermostable coiled coil we used to assemble the enzyme appears to lend it stability. We will examine whether coiled coil-mediated assembly of proteins into cages is a generally effective method to stabilize enzymes. We will investigate whether this can be achieved by incorporating other trimeric enzymes into similarly constructed icosahedral cages. We will further extend our investigation by examining the effectiveness of other cage geometries for stabilizing enzymes. Construction of enzyme nanoreactors We consider that our assembly strategy may be particularly well suited to developing enzyme nanoreactors that could incorporate two or more enzyme activities for the catalysis of reaction cascades. For this application stability against thermal and chemical denaturants would be very useful. Our approach allows us to incorporate one enzymatic activity as part of the cage wall. We will explore strategies for elaborating deisnged protein cages with additional catalytic domains and for encapsulating enzymes within these cages. This potentially allows the construction of spatially controlled multienzyme complexes that could efficiently catalyze sequential reactions involving reactive or short-lived intermediates.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 09, 2020
- Source ID
- W911NF2010150
Entities
People
- Neil Marsh
Organizations
- Army Contracting Command
- United States Army
- University of Michigan